An Infrared Study of CO Binding to Heart Cytochrome c Oxidase and Hemoglobin A IMPLICATIONS RE O2 REACTIONS*

(Received for publication, February 22, 1977)

SHINYA YOSHIKAWA,$ MILES G. CHOC, MARY C. O’TOOLE,~ AND WINSLOW S. CAUGHEYI

From the Department of Biochemistry, Colorado State University, Fort Collins, Colorado 80523

The CO stretch bands for CO liganded to hemoglobin A (1951 cm-l) and to fully reduced cytochrome c oxidase (1963.5 cm-‘) were used to determine the amount of CO bound, to follow the exchange of CO from the oxidase to Hb, and to probe the nature of the CO (and 0,) binding sites. A variety of oxidase preparations from bovine heart were ex- plored; a convenient route to high purity oxidase at the concentrations (>I.5 mu) convenient for infrared use was developed. An emu (1963.5 cm-l) of 4.9 * 0.3 for the oxidase carlnmyl and an eW (1951 cm-l) of 3.7 f 0.1 for HbCO were found; both extinctions were independent of concentration. Although the oxidase band (Av,,~, 6 cm-‘) is narrower than the Hh band (Av,,~, 8 cm-‘), the areas (integrated intensities) are comparable (28 and 34 rnn-’ cm-2, respectively). The affinity of I-Ib for CO is -56 times greater than the oxidase (Kd = 0.3 CM in 0.65 M sodium phosphate buffer, pH 7.4, 24”). The CO to heme A stoichiometry was established as 0.5 in several types of experiments. This CO/Fe ratio appeared insensitive to the degree of purification or to the activity of the enzyme and demonstrated that only one metal (the classical a3 heme) of the four possible sites (2 Fez+, 2 Cd+) is liganded by CO under these conditions. OS is reasonably assumed to bind at the same site to form an iron oxy- genyl species as in I-IbOz. The exceedingly narrow oxidase carbonyl band (the narrowest yet seen for a hemeprotein car-bony!) indicates the CO ligand is not exposed to the aqueous medium. Rather it is in a highly ordered environ- ment that is presumably largely nonpolar (hydrophobic). The infrared parameters (frequency. width, intensity) for CO show the oxidase site to be more like the Hb sites than the sites of other hemeproteins (e.g. cytochrome P-450, cytochrome P-420, and peroxidases). This isolation from aqueous medium is important to the mechanism of 0, reduc- tion, e.g. by stabilizing neutral species over charged species. Since CO binds to only one heme A, the data do not provide information about the likely second metal (copper or iron) in the formation of a suggested p-peroxo intermediate dur-

* This work was suuwrted bv United States Public Health Service Grant HL-15980. -- -

$ Permanent address, Department of Biology, Konan University, Kobe, Japan.

li Present address, Department of Chemistry, Briar Cliff College, Sioux City, Iowa.

ll To whom correspondence should be addressed.

ing the 0, reduction process. These data do demonstrate the considerable utility of infrared techniques to directly probe ligand binding quantitatively as well as qualitatively.

Cytochrome c oxidase, the copper hemeprotein of the inner mitochondrial membrane, is the site of major oxygen utiliza- tion and an associated energy coupling for oxidative phospho- rylation (1). The structures of heme, protein, and lipid compo- nents and the physical properties, as well as the mechanisms for oxygen reduction associated with this oxidase, are thus of considerable current interest (l-3). Beginning with the classi- cal studies of Warburg (4) and Keilin and Hartree (5) on effects of carbon monoxide on respiration, studies which led to the discovery and early characterizations of cytochrome c oxidase, carbon monoxide has represented an important inhibitory probe for the ox&se as it has for hemoglobin and many other hemeproteins. Since it is reasonable to assume that CO com- petes with 0, for a common binding site, a better understand- ing of CO binding is expected to also provide insight re 0, binding.

Infrared spectroscopy provides an effective direct probe for CO binding to hemes and to hemeproteins (within, or isolated from, intact tissue) (6-8). For hemoglobin A carbonyl the absorption band at 1951 cm-’ is due to bound CO (6). With the carbony complex of fully reduced oxidase, Caughey and co- workers found the C-O stretch band at 1963.5 cm-’ (9-11). This frequency is near that observed for other hemeproteins and protein-free heme carbonyls but is about 100 cm-’ lower than the frequencies noted for Cu(1) carbonyls (e.g. the hemo- cyanin carbonyl (12, 13)). Thus, CO appears bound to Fe(D), and not to Cu(I), in the oxidase. Furthermore, the infrared band widths, if suitably interpreted, can provide information on the nature of the environment immediately around the ligand. The infrared technique may also be used for the quan- titative evaluation of how much CO (or other ligand) is bound to a hemeprotein (11, 14, 15).

We report here the use of infrared CO band intensities to quantitate the extent of CO binding to hemoglobin and to the oxidase. Implications re 0, reactions that may be drawn from a consideration of the infrared data for carbonyls are dis- cussed. CO binding to a variety of oxidase preparations was explored; a method for isolation of the oxidase from bovine heart preliminarily reported by Volpe and Caughey (16) is

5498

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Infrared Studies of CO Binding to Cytochrome c Oxidase and Hb 5499

described in detail. Brief accounts of some of the infrared studies have also appeared (14, 15, 17).

MATERIALS AND METHODS

Activities were determined spectrophotometrically (18) in 0.1 M sodium phosphate buffer, pH 6.0, at room temperature (24 -C 1”). The assay was initiated by adding 10 ~1 of oxidase solution about 1 FM in heme A to 3 ml of buffer solution containing 15 PM cytochrome c (Sigma type VI) in a l-cm path length cuvette. Absorbance changes were monitored at 550 nm and rates are expressed as seconds-‘/mg of protein/3 ml.’ Extinction coefficients for the infrared CO bands, e.g. l ,,(1951 cm-‘) and l ,M(1963.5 cm-‘), are expressed in units of con- centration’ path length-’ (mr+-’ cm-i), where the concentration represents the concentration of CO bound, and not necessarily the concentration of heme present.

Iron analysis was performed by the method of Doeg and Ziegler (19) with the reagents prepared and the glassware handled as sug- gested by van De Bogart and Beinert (20). Iron was quantitated as the bathophenanthroline (Sigma) complex at 535 nm; the mercapto- acetic acid was purchased from Eastman and the “Dilut-it” analyti- cal iron standard from J. T. Baker. Copper was quantitated as the bathocuproin (Sigma) complex at 479 nm (21). For some prepara- tions, copper/iron ratios are determined by the Analytical Chemistry Facility, Colorado State University whereby the samples were plasma-ashed under vacuum, taken up to final volume in 1:l (v/v) nitric acid, and analyzed for iron and copper on the atomic absorp- tion spectmphotometer.

Ammonium sulfate was purchased from Schwa&Mann (enzyme grade). Bovine heart was generously donated by Monfort of Colo- rado, Inc. Oxyhemoglobin was isolated from fresh human blood by the method of Geraci et al. (22). Sodium dithionite was purchased from Fisher Scientific Co. and sodium cholate from Sigma.

Recording of Infrared Spectra

Infrared spectra were recorded with a Perkin Elmer model 180 spectmphotometer in the absorbance mode under dual beam opera- tion at a scan rate of 3 cm-‘/min, a resolution setting of 2.5 cm-’ at 2900 cm-’ (best results were obtained when the resolution sAu,,,l2 at the absorbing frequency*), a 20-fold ordinate expansion, and a time constant of 3. Cells contained calcium fluoride windows. The sample cells had a path length of 0.055 mm whereas a variable path length cell containing water was generally used for the reference cell. The base-lines of the carbonyl bands used to measure peak heights and areas were drawn through the points on each side where the band was first clearly detected to rise. The visible spectrum of the sample was frequently examined before or after recording the infrared spec- tra, or both, by placing the infrared cell directly in the sample compartment of a Gary 17 spectrophotometer.

Determination of Carbonyl Stretching Band at Various HbCO Concentrations

The concentration of HbO, in 0.01 w sodium phosphate buffer, pH 7.4, was determined from an a-band l mM of 15.16 (23). HbO, solutions with concentrations from 0.2 to 3.0 mxr in heme were saturated with CO gas at room temperature. About 0.2 ml of the HbO, solution in a 3-ml syringe was flushed three times with CO, allowed to stand under CO atmosphere for 20 min, and then forced into an infrared cell. Infrared spectra were recorded from 2000 to 1900 cm-‘. The area or the peak height at 1951 cm-‘, or both, was measured.

Titration of Oridase(0) with CO by Visible Difference Spectroscopy

A solution of known CO concentration was prepared by passing CO gas through 0.01 or 0.05 M sodium phosphate buffer, pH 7.4, for 30 min. adding a small amount of sodium dithionite to remove oxygen, sealing the bottle with a rubber septum, and blowing CO over the surface for 10 min. A rubber balloon was connected to the

’ Protein amount is actually based upon iron analysis, assuming 10 nmol of heme A/mg protein.

* The abbreviations used in this paper are: Av,,,, the band width at half-peak height in absorbance; Oxidase(O), fully reduced oxidase; Ox&se(W), fully oxidized oxidase; Oxidase(1 . . . III), oxidation- reduction states between the fully reduced and fully oxidized en- zyme, in which the Roman numeral signifies the number of electrons removed from the fully reduced state.

vessel via a needle through the septum to permit the maintenance of a slight positive pressure above the solution while milliliter amounts of buffer were removed from the sealed vessel (200 ml). The concen- tration of CO in solution was determined from the infrared spectrum for a mixture of the CO solution with about 10 mru Hb solution (4:1, v/v); the CO stretch band intensity (either height or area) for the HbCO produced was used to compute the amount of CO present.

A solution (1 ml) of oxidase (Preparation A), about 0.1 rnhf in heme A, which had been reduced by exposure to excess dithionite for 20 min was transferred to quartz cells with 5-mm path lengths and sealed with rubber septum caps, and nitrogen gas was blown over the surface for 10 min. Difference spectra from 700 to 510 nm were recorded on a Cary 17 spectrophotometer at 24 f l”, after lo- or 20-~1 aliquots (Hamilton microsyringe) of CO solution were successively added to the sample cell; N,-saturated buffer was similarly added to the reference cell. The peak to trough absorbance difference (A,,, nm -A aor& was monitored as the CO concentration increased. The stabilities of both Oxidase(0) and Oxidase(0). CO were examined by allowing them to stand under experimental conditions for 2.5 h (more than twice the time required for the experiment) after which, the absorbance at 605 nm decreased by only 1.5%.

Infrared Determination of HbCO Formed from Oxidase(0) ‘CO and HbO,

A solution of Oxidase(IV) (0.25 ml, 1.0 to 1.6 mru, Preparations A or B) in a 3-ml syringe to which was added a slight excess of solid sodium dithionite was flushed twice with 3 ml of CO gas, and then exposed to a final 3 ml of CO at 4” for 1 h while the syringe was being rotated slowly at an angle of about 30” from the horizontal position to allow more surface exposure to the CO atmosphere. Excess CO gas was expelled completely by forcing the solution to a point flush with the end of the syringe. Then, 0.125 ml of about 9 to 19 rnM HbOt was injected into the syringe which was then shaken gently for 30 s to mix the contents. The mixture was transferred to an infrared cell and the spectrum from 2000 to 1900 cm-’ was recorded. Blank deter- minations of CO concentrations were made on CO-saturated buffer solutions to which no oxidase had been added.

Determination of CO Present in Mixtures of Oxidase(0) and Hb by Infrared Spectroscopy

Preparation of Solutions ofOridase(0) Saturated with CO -First, 1.05 ml of 1.2 to 1.6 mM Oxidase(IV) in 0.01 M sodium phosphate buffer, pH 7.4, was placed in a glass bottle (2.2 cm in a diameter and 3.6 cm high) that was sealed with a serum cup and cooled in an ice water bath. A venting needle was inserted through the cap and CO gas was then blown gently via a needle over the surface of the liquid for 25 min to deaerate the system. Solid sodium dithionite (5 mg) was quickly added. Passage of CO through the system was continued for an additional 40 min with occasional gentle shaking of the bottle. A balloon with about 100 ml of CO gas was attached via a needle to the bottle which was rotated slowly for 60 min. The contents were then sampled for infrared experiments as described above. Standing for as long as 5 h (at 0”) was observed to cause no significant changes in the infrared spectrum of Oxidase(0) CO.

Infrared Spectra of Oxidase(O)~CO Solutions with Varying Amounts of Added Hb -A test tube (9 x 30 mm) with solid sodium dithionite and a serum cap seal was flushed with N, gas via entry and exit needles for 5 min. The amount of dithionite was slightly in excess of that needed to render anaerobic a solution prepared by addition, via a microsyringe, of 20 to 60 ~1 of HbO, (about 7 mru in heme) in 0.01 M sodium phosphate buffer, pH 7.4. The tube was shaken gently and examined to ascertain that all the dithionite was dissolved, then 0.125 ml of CO-saturated Oxidase(0) solution pre- pared above (see “Preparation of Solutions of Oxidase(0) Saturated with CO”) was added via a l-ml syringe. The contents were gently mixed then transferred via a 3-ml syringe to an infrared cell and the spectra were recorded from 2000 to 1900 cm-‘. Exposure of the solu- tion to oxygen during the transfer was minimized by prior flushing of syringe and cell with N, gas.

Isolation of Cytochrome c Oxiakse from Bovine Heart uia Modification of Method of Volpe and Caughey (16) (Prepamtion A)

Heart Particle Preparation -The muscle of bovine heart (1.8 kg) was freed of fat and connective tissue and passed through a meat grinder. Two 900-g portions of the resulting mince were washed by suspension in 20 liters of deionized water, collected on cheesecloth in

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5500 Infrared Studies of CO Binding to Cytochrome c Oxiokse and Hb

a large Buchner funnel, squeezed in the cheesecloth to about the original volume, combined with 2.4 liters of 0.04 M sodium phosphate buffer, pH 7.4, and 230 g of crushed ice (prepared from glass distilled water) in a 4-liter Blendor (Waring), and homogenized at medium snead for 3 min. All subseauent operations were carried out near 4”. The homogenate was cen&fuged at 2500 rpm (IEC model UV centri- fuge with head No, 976. l-liter bottles) for 40 min, and the supema- Gt was saved. The precipitate (1.5 to 2.0 liters), combined with 1.2 liters of the same buffer and 230 g of ice, was rehomogenized at medium speed for 3 min. Centrifugation as before yielded a precipi- tate that was discarded. The supematants from the two centrifuga- tions were combined. adjusted to pH 5.4, with 1 M acetic acid, and centrifuged as above: The precipit& was suspended in 200 ml 0.2 M sodium ohosphate buffer, pH 7.4, with a Potter-Elvehjem homoge- nizer, then adjusted to pH 7.4 with 3 N NaOH, and allowed to stand overnight at 4”.

Fractionation in 3% Cholote -A particle suspension with an equal volume of particles and 0.2 M sodium phosphate buffer, pH 7.4 (tvnicallv 575 ml. nreuared from above by adding -90 ml of 0.2 M __ sodium phosphambuffer, pH 7.4), was treated with 50 ml of 40% sodium cholate in 0.1 M sodium phosphate buffer, pH 7.4, and then adjusted to 33% saturation in ammonium sulfa&The ammonium sulfate was added immediately after the addition of cholate with concomitant addition of 3 N NaOH to maintain the pH at 7.4 during addition of salt (the pH was controlled in this way in all subsequent additions of ammonium sulfate). After standing for 25 min with stirring, the mixture was centrifuged for 20 min at 17,000 rpm (IEC Centrifuge model B-20A, head No. 870; this head was used in all subsequent centrifugations). The supematant was adjusted to 50% saturation in ammonium sulfate and centrifuged for 20 min at 17,000 wm.

Dmalysis -The precipitate was dissolved (stirring with a glass rod) in 220 ml of 0.1 M sodium phosphate buffer, pH 7.4, containing 0.5% sodium cholate. The solution was dialyzed-against 0.04 M sodium phosphate buffer, pH 7.4, for 90 min and the partially precipitated preparation was then centrifuged for 90 min at 19,000 rpm.

Fmctionations in 2% and 0.5% C&lute -The precipitate was sus- pended in 200 ml of 0.1 M sodium phosphate buffer, pH 7.4, contain- ing 2% sodium cholate with a Potter-Elvehjem homogenizer. The suspension was brought to 25% saturation in ammonium sulfate, was allowed to stand for 30 min, and was then centrifuged for 10 min at 17,000 rpm. The supematant (210 ml) was brought t.n 45% satura- tion in ammonium sulfate and centrifuged for 10 min at 17,000 rpm. The precipitate was suspended in 200 ml of 0.1 M sodium phosphate buffer, pH 7.4, containing 0.5% cholate, and the suspension was adjusted to 25% saturation in ammonium sulfate. After standing for 30 min. the turbid solution was centrifuged for 10 min at 17,000 rpm. The supematant (200 ml1 was adjusted to 40% saturation in ammo- nium sulfate and centrifuged for 10 min at 17,000 rpm. The precipi- tate was dissolved in 130 ml of 0.1 M sodium phosphate buffer, pH 7.4, containing 0.5% cholate (A553nmlABOsnm = 0.53; ASaUnm/ABOSnm = 0J~51.~ The clear solution was adiusted to 25% saturation in ammo- nium sulfate, was allowed to stand for 30 min, and was then centri- fuged for 10 min at 17,000 rpm. The supematant (140 ml) was ad$.mted to 35% saturation in ammonium sulfate and centrifuged for 10 min at 17,000 ‘pm.

Fmetionutions in 0.75% and 0.5% Tureen 20 -The precinitate was dissolved in 200 ml of 0.1 M sodium phosphate buffer, pH 7.4, con- taining 0.75% Tween 20 (A,,,.,/A ROQnm = 0.411.” The solution was adjusted to 25% saturation in ammonium sulfate and centrifuged immediately for 10 min at 17,000 r-pm. The supematant (210 ml) was adjusted to 35% saturation in ammonium sulfate and centrifuged for 10 min at 17,000 rpm. The precipitate was dissolved in 80 ml of 0.1 M sodium phosphate buffer, pH 7.4, containing 0.5% Tween 20 (A,,.,/

A - 0.34; AJBOnmlA~s,,,,, = 0.37La 805”nl - The solution was adjusted to 25% saturation in ammonium sulfate

and centrifuged for 10 min at 17,000 rpm.4 The supematant (80 ml) was adjusted to 35% saturation in ammonium sulfate and centri- fuged for 10 min at 17,000 rpm. The precipitate was dissolved in 80 ml of 0.1 M sodium phosphate buffer, pH 7.4, containing 0.5% Tween 20

3 Typical absorbance ratios after reduction with excess dithionite and standing for 20 min.

* When Twesn 20 is present, firm pellets were not always ob- tained. Some precipitate floated or adhered to the sides of the centri- fuge tube, or both. In these instances the supematant was separated by decanting or filtration through paper (Whatman No. 1).

and the solution was adiusted to 33% saturation in ammonium sulfate. The precipitate, obtained from centrifugation for 10 min at 17,000 rpm, was dissolved in 80 ml of 0.1 M sodium phosphate buffer, pH 7.4. After the solution was brought to 33% saturation in ammo- nium sulfate, it was centrifuged for 10 min at 19,000 rpm to give a precipitate that was dissolved in 0.01 M sodium phosphate buffer, pH 7.4.

Dialysis and Concentration-The solution was dialyzed against the same buffer for about 12 h. The supemamnt (0.3 to 0.5 mM in heme A) from centrifugation for 20 min at 19,000 rpm was concen- trated to about 2 mM heme A in an Amicon “Diaflow’ apparatus. To achieve a concentration of 1 mM, 4 or 5 h were required; to reach 2 mM required more than 10 h. For the best stability, at least 1 mM concentrations appeared optimal. Yields of from 7 to 10 pmol in heme A/preparation were obtained in an overall elapsed time of 3 days.

Isolation of Cytochrome c Oxidase from Bovine Heart via Modification of Method of Okunuki et al. (24) (Preparation B)

Heart Particle Preparation -The muscle of bovine hearts was freed of fat and connective tissue and nassed through a meat grinder to give 1.8 kg of mince. Each half (900-g port&l was placed in a Waring Blendor along with 300 ml of 0.2 M sodium phosphate buffer, pH 7.X and 1.8 liters of deionized water at 4”. The mixture was homogenized for 3 min at medium speed. The homogenate was centrifuged (IEC Centrifuee model UV. head No. 976: l-liter bottles)

-~9-~ for 40 mm at 2500 rpm. The precipitate was placed’in the blender with 150 ml of the same buffer and 1.36 liters of cold water. The mixture was homogenized and centrifuged as before. The four super- natants were treated as in the Preparation A described above.

Fractionation in 2.5% Cholute -The particle suspension was ad- justed to contain 2 volumes of 0.2 M sodium phosphate buffer, pH 7.4, per original volume of particles as well as s/a volume of 2.5% sodium cholate plus i/s volume of distilled water and was then brought to 25% saturation in ammonium sulfate (in this procedure the pH was maintained at 7.4 during the sulfate addition by additions of 28% ammonium hydroxide). After standing overnight, the mixture was brought to 35% saturation in ammonium sulfate, was allowed to stand for 3 h, and was centrifuged as above.

Subsequent Steps -The supematant brought to 50% ammonium sulfate gave a precipitate which was successively subjected to two fractionations between 25 and 36% ammonium sulfate in the pres- ence of 2% cholate, to four fractionations between 25 and 35% ammo- nium sulfate in the presence of 1% cholate, and to three fractiona- tions between 25 and 33% ammonium sulfate in 0.25% Tween 20. The final precipitate from these fractionations was dissolved in 0.01 M sodium phosphate pH 7.4. The solution was dialyzed against the same buffers overnight and then concentrated to -2 mM in heme A as in Procedure A. Yields of from 15 to 20 mmol in heme A/prepara- tion were obtained in an overall elapsed time of 4 days.

RESULTS

Physical Properties of Oxidase Preparations A and B- Properties of oxidase Preparations A and B are listed in Table I. The two preparations differ most strikingly in their activi- ties. These differences may result primarily from longer expo- sure to cholate in the Okunuki procedure (B); others have suggested that exposure leads to deactivation of the oxidase (25, 26). The copper to iron ratios are typically only slightly greater than one for each preparation. In contrast to findings reported earlier in Volpe and Caughey (161, the extinction coefficient of the reduced a-band for our A-type preparations was not affected by the presence or absence of Tween 20 in the buffer; for the case where no Tween 20 was added, the value observed here is approximately 30% greater than was reported in the earlier study.5 However, the presence of 0.5% ‘I’ween 20 was seen to increase the activity of both preparations; an increase of nearly 160% was noted for an A-type preparation. Spectra for the visible and Soret regions of both preparation,

5 The reasons for different findings in the earlier study remain unclear.

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Infrared Studies of CO Binding to Cytochrome c Oxio!use and Hb

with or without CO, appear identical with no significant dif- ferences in extinction coefficients as can be noted in Figs. 1,2, and 8. The CO stretch of fully reduced enzyme was always at 1963.5 cm-‘; extinctions were essentially the same for all preparations.

Determination of CO Concentration via Infrared Band at 1951 cm-’ for HbCO -A method that is frequently used to prepare buffer solutions with known concentrations of CO is to saturate the buffer with CO gas at a specific temperature and pressure and then to use a previously determined solubility of CO in water to ,estimate the amount of CO present (27). We developed a direct approach in which the HbCO infrared stretch at 1951 cn-I is used to quantitate the CO present in a particular buffer solution. The buffer solution of unknown CO concentration is mixed with a 3- to 4-fold molar excess of -9

TABLE I Properties of oxiduae preparations A and B

Preoamtion A B

Time elapsed during puri- fication and concentra- tion (days)

Yield (g) Activity (s-‘/mg protein/3

ml) ~i~(%~ nm (mr.-’

CID-=‘JC Oxi~eKOAeo,,40 nm

(rnt.-l cm-9’ Oxidase(0) CO c,~.~ cm-i

(mM-l inn’) Qxidase(O)h,,. (nmP Oxidase(IVh,., (nrn)” Cu:Fe

3

0.7-1.0” 1.5-2.00 11.7 f 2.06 1.6-2.6

19.9 + 0.5

17.6 r 0.3

4.9 2 0.3

606, 565, 516, 444 598, 419

1.15 f 0.04

4

20.7 z 0.7

4.9 k 0.4

605, 565, 516, 444 598, 419

1.22 + 0.07

* From 1.E kg of heart muscle mince. b Occasionally, significantly bigher or lower activities were ob-

served for reasons still not well understood. r Concentration of heme A in 0.01 M sodium phosphate, pH 7.4. d Reduced with slight excess ditbionite. e As obtained from purification procedure.

01 I I I 400 600 IO

WAVELENGTH (nm)

FIG. I. Electronic spectra of cytochrome c oxidase (CCO) @‘repa- ration A) in 0.01 M sodium phosphate buffer, pH 7.4. -, Oxidase(O) reduced with excess dithionite band maxima in nanometers &J: 60.5 (19.9), 665 (8.1), 518 (7.1). 444 (107). ---, OxidsseUV) as isO- Iated, fully oxidized, band maxima in nanometers (e,.): 598 (8.7), 419 (82).

mM HbO,. The binding of dissolved CO by Hh is essentially complete. When this solution is examined in the infrared over the frequency range 2000-1900 cm-‘, only a band at 1951 cm-l due to CO bound to Hb is observed; excess HbO, does not contribute any absorbance to this band (the O-O stretch appears at much lower frequency, 1106 cn-’ (28)). Either the height or the integrated intensity of the C-O band may be used to determine the amount of CO present. The integrated intensity is 34 rnMP cm-*. Fig. 3 shows a least squares tit of a set of data points generated by plotting the band heights (in centimeters) at 1951 cm-’ as a function of known HbCO con- centrations (0.2 to 3.0 mM); fi-om these data an emM value of 3.7 is computed. This plot represents a typical finding of linearity and high precision for such intensity versus concentration plots. Such an extinction coefficient permits the rapid and accurate determination of the amount of dissolved CO in an unknown solution whether or not the solution is saturated with CO.

Titration of Oxio!ase(O) with CO followed by Visible Differ- ence Spectroscopy -The detection of subtle ligand-induced spectral changes by difference spectroscopy has been a classi-

\ \ EcCoO I%co - _--_---

60- :, ; I

-40

: : I :

A

I O 400

I 1

WPEJELENGTH hii?

0

FIG. 2. Electronic spectra of cytochrome c oxidase (CCO) Prepa- ration A) in 0.01 M sodium phosphate buff’er, pH 7.4. -, OxidaMO) with excess ditbionite. - - -, Oxidase(0). CO band maxima in nano- meters (e,,,& 603 (19.6). 590s (15.2), 551 (10.3), 517 UO.l), 432 (66).

16 -

I 1 1 I I 1 0

Hb-% CONCENTRATlO?(mM) 3.0

FIG. 3.* plot of C-O stretch band height at 1951 cm-’ versus HbCO concentration. Solutions of known concentration of Hb in 0.01 M sodium phosphate buffer, pH 7.4, were saturated with CO and the C-O stretch band height was measured in centimeters.

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5502 Infrared Studies of CO Binding to Cytochrome c Oxidase and Hb

cal technique for the observation of ligand binding. The spec- tral changes in the visible region induced by CO binding to Oxidase(0) monitored in this way appears in Fig. 4 wherein isosbestic points can be observed at 598, 577, and 566 run. Titrations performed on four separate enzyme preparations (Preparation A, 0.948 to 0.197 nnu heme A> gave l mM, 590-80,nm = 4.6 f 0.4 which compares with the +,9-n,,, = 4.5 reported by Vanneste (29). A plot of the peak to trough absorbance (59o-607 run) uers’sus CO concentration as in Fig. 5 can be used to calculate the concentration of CO binding sites. In Fig. 5, the data points give a best tit to a theoretical binding curve based upon 56.8 PM CO binding sites, 107 pM heme A, and an Oxidase(0). CO dissociation constant, Kd = 0.30 PM. The CO to iron stoichiometry computed from four experiments was 0.47, 0.41, 0.43, and 0.47 CO/Fe. The arrows in Fig. 5 indicate the positions of the endpoints expected for CO/Fe = 0.5 and 1.0. Clearly not all of the iron forms a complex with CO; rather, only about one-half of the iron binds CO. Recently, Wharton and Gibson (27) briefly reported similar results; in one experi- ment they found 2.8 PM sites for 6.3 ELM heme A and a Kd of 6

X lo-’ M and in a second experiment, 36 PM sites for 63 PM

heme A. Earlier attempts by others (30) had yielded somewhat lower ratios. In general, experiments of this type have yielded CO/Fe ratios significantly less than 0.5. To be sure, these particular experiments do not exclude the possibility of there being another binding site of reasonably high affinity which causes no visible spectral perturbations but, as will be shown, the infrared experiments give no indication of such a second site.

CO Exchange from Oxio!use(O)~CO to Hb followed by In- frared Spectrosco~ -As noted above, the infrared stretching frequencies for CO bound to Oxidase(0) and Hb were found at 1963.5 and 1951 cm-‘, respectively, and these absorption bands corresponded only to CO bound to the heme iron. This fre- quency separation (hv = 12.5 cn-‘1 is great enough to allow for the simultaneous observation and quantitation of the two bands due to the presence of both Oxidase(0) * CO and HbCO in one sample mixture (Fig. 6). Upon the sequential addition of a

1 [CCOXO]= 1.45 mM A

[co]= 0.94 mM [*.col.o h P

.a

1 520 600 700

WAVELENGTH (nm)

FIG. 4. Visible difference spectra generated on titration of Oxi- da&O) with CO in 0.05 M sodium phosphate buffer, pH 7.4, at 26 + 1”. Ten-microliter aliquots of buffer with 0.99 rnM CO were added sequentially to the sample cells; equal amounts of N,-saturated buffer were added concomitantly ta the reference cell. The cells (quarts, with 5mm path length) were sealed by rubber septum caps to prevent oxygen contamination.

1 I 1 I I 20 40 60 60 IO0 120

CO CONCENTRATION (ph4)

FIG. 5. Titration of Oxidase(0) by CO buffer. Experimental condi- tions were those given in the legend of Fig. 4. The solid line is a theoretical binding curve based on 107 pM heme A, 59.8 /.LM CO binding sites, and a CO dissociation constant for Oxidase(0) CO: K* = 0.30 PM. The arrows indicate the end point positions for expected CO/Fe smichiometries of 0.5 and 1.0.

lccocol= 1.45mM 6

FREPUENCY kr~i”)

FIG. 6. Exchange of CO from Oxidase(0) to Hb (see “Materials and Methods”). Increments of Hb 0, (9 rnd solution were added to a solution with 125 ~1 of Oxidase(O).CO (1.68 mM heme A) and satu- rated with CO. Small additions of Hb caused formation of HbCO (detected at 1951 cm-‘) by reaction with CO free in solution. Larger additions of Hb reduced the 1963.5 cn-’ band height while the 1951 cm-’ band increased until the additions were large enough for all the CO on the oxidase to be transferred to Hb. All concentrations are corrected to 145 ~1 total volume. A, 125 ~1 of Oxidase(0) . CO + 20 ~1 of H,O; B, 125 ~1 of Oxidase(0). CO + 20 ~1 of Hb; C, 125 ~1 of Ox&se(O). CO + 25 ~1 of Hb, D, 125 ~1 of Oxidase(0) . CO + 28 ~1 of I-& E, 125 yl of Oxidase(O).CO + 30 ~1 of Hb. CCO, cytochrome c oxidase.

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Infrared Studies of CO Binding to Cytochrome c Oxidase and Hb

Hb solution (>9 mr& to a solution of Oxidase(O).CO (-1.5 mM heme A), the 1951 cm-’ band intensity increased in direct proportion to the amount of added Hb. Thus, essentially all the Hb forms a complex with CO until all the CO has become bound to Hb. Initially, no change in the 1963.5 cm-l band height is observed when a quantity of Hb less than, or equal to, the amount of free CO is added. Subsequent Hb additions result in a decrease of the 1963.5 cm-’ band concomitant with a further increase of the 1951 cn-’ band. The ratio of the change in band height at 1963.5 cm-’ to the change in band height at 1951 em-’ can be used to compute the extinction coefficient of the 1963.5 cm-’ %and from the known HhCO extinction value (above).

In Fig. 7 the band height at 1963.5 cm-’ is plotted uersus band height at 1951 cm-‘. The initial increase in the 1951 cm-’ band, while the 1963.5 cm-’ band remains constant, corre- sponds to the titration of the free CO by Hb. The portion with negative slope = 1.41 then corresponds to the exchange of CO from Oxidaae(0) to Hb. The slope itself corresponds to the ratio of extinctions of the 1963.5 cn-’ and 1951 cm-l bands. Thus, Ox&se(O) with 1 mM CO bound will have a 1963.5 cm-’ band intzm&y 1.4 times greater than the 1951 cm-’ band intensity of Hb with 1 mM CO bound, whereas the two bands will exhibit nearly identical areas (integrated intensities). Such Oxidase(0) * CO and HbCO bands integrated by either plani- meter or triangulation methods have been examined many times with a uniform result that little difference in integrated intensities between the two proteins was observed. The CO/Fe ratio for Oxidase(0) * CO can be determined from the height of the 1963.5 cm-l band in a solution of known heme A concentra- tion. Three independent exchange experiments., in each case an A-type preparation, gave E,,,~, 1963.5 cm-’ values of 4.8,4.8, and 5.2 and CO/Fe values of 0.47, 0.51, and 0.49. In this method of computation, the concentration of CO free in solu- tion is assumed to remain constant (and very small) during the exchange of CO from Oxidase(0) to Hb. The method is therefore not dependent upon the relative affinities of Oxi- da&O) and Hb for CO but the much higher affinity G-50 times) of Hb (K, = 4.6 nM, calculated from F&f. 31) allows Hh t.0 compete with the oxidase for CO very effectively.

We have also carried out exchange experiments with both Freparations A and B by the method described by Volpe et al. (11). A large excess of HbOz was added to an Oxidase(0) solution (1 to 1.6 mM) that had been saturated with CO gas (the concentration of heme B was always greater than three

BAND MIGHT km) AT 1951 cm-’

FIG. 7. Plot of band heighta at 1963.5 cm-’ ucrs~s 1951 cm-’ obtained upon the incremental addition of Hb to Oxidase(0). CO as shown in Fig. 6. Band heights are corrected to a total volume of 145 ~1. The slops, 1.41 = •,~.~ cm-l/~18J, o+.

times that of heme A). The total CO present was determined from the intensity of the HbCO band at 1951 cm-‘. The amount of dissolved unbound CO in the oxidase solution, which was estimated by carrying out the experiment in the absence of oxidase, was used as a blank in the calculation of oxidase- bound CO. The CO/Fe ratios found for 21 determinations with Preparation A and 28 for Preparation B are 0.48 + 0.10 and 0.46 f 0.07, respectively, i.e. about one-half that reported previously (ll), and are fully consistent with one CO binding site/2 heme A molecules in the oxidase. Visible spectra for CO complexes of both preparations obtained directly from infrared cells are shown in Fig. 8 to demonstrate that visible as well as infrared spectra can be obtained on the same sample and that the spectra for the two preparations are similar.

DISCUSSION

Determination of CO by Infrared Spectroscopy--The find- ings reported here demonstrate that infrared spectroscopy is an effective technique for the direct quantitative determina- tion of CO bound to hemeproteins. Either the peak height or the integrated intensity (area) of the CO stretch band can be measured. However, the peak height is more easily obtained. The extinction coefficients (from peak heights) for HbCO and for Ox&se(O). CO were found to be constant over the concen- tration ranges studied as required for an accurate and precise method for the determination of CO. The infrared technique is applicable to protein, and even intact tissue, under physiologi- cal conditions (e.g. body temperature, neutral pH, and aqueous medium) and to CO concentrations in the range 0.01 to 10 mM, a range within which many hemeproteins are found in tissue.

Stoichiometry of CO Binding to Cytochrome c Oxiokzse- That, in fact, 1, and only 1, CO will bind per oxidase molecule of 2 hemes and 2 coppers is convincingly supported by the several lines of evidence presented here. Furthermore, the extent of CO binding was not found sensitive to the stage of purification, the method of isolation, or the activity of the enzyme. Such a stoichiometry is, of course, fully consistent with the classical suggestions of Keilin and Hartree that there are two spectrally and functionally distinct heme A moieties (a and a& with only the aQ heme capable of reaction with CO (or

FIG. 8. Visible spectra of Oxidase(O).CO taken with sample in infrared cells with CaF, windows and a path length of 0.056 mm. Preparation A, 1.48 rnM heme A (- - -); Preparation B, 2.02 mx heme A t-j.

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5504 Infrared Studies of CO Binding to Cytochronw c Oxiduse and Hb

0,) (5). Indeed CO/Fe ratios that approach 1:2 have been seen in electron transport particle preparations (29, 32). However, for purified preparations lower CO/Fe ratios have generally been found (e.g. 1:4 to 1:2.5 (321, 1:3.8 (301, and 1:2.4 (2911, which may be interpreted as indicative of more than 2 hemes in the minimum functional unit of the enzyme. Suggested possible causes of these low values have included the incom- plete reduction of a,, the incomplete saturation with CO, and the rigors of purification resulting in a lesser amount of redu- cible a, in purified preparations (29, 30, 32). On the other hand, ratios greater than 1:2 have been reported for prepara- tions with a&red Soret spectral characteristics (29). In a preliminary report, Volpe et al. (11) suggested a CO/Fe ratio of 1:l for a preparation that contained no apparent spectral ab- normalities; however, their reported value for the integrated absorption intensity of the CO stretch band does not agree with values for several other hemeprotein carbonyls we have obtained (33). The consistency we observed for such values caused us to seriously question the Volpe et al. (11) finding scnm after its appearance. And, aRer the present work was well underway, the accuracy of the I-lb exchange experiments used by Volpe et al. was questioned in the brief communica- tion by Wharton and Gibson (27); we have also noted the variability of the exchange experiments. In our attempts to duplicate the Volpe et al. experiments wherein exchange of CO from Ox&se(O) + CO to Hb was followed by infrared spec- troacopy, we found much more CO (about 56% more) dissolved in the buffer, a value important as the blank determination. Since the in-solution and heme-bound species of CO are pres- ent in approximately the same amounts, this discrepancy can account for the difference in computed stoichiometries. We also found the integrated absorption intensity for Oxi- da&O) .CO is nearly identical to values found for HbCO and other car-bony1 hemeproteins. However, it does not seem possi- ble, or even worthwhile, at this time to attempt further expla- nation for the deviations from a CO/Fe ratio of 0.5 that have been reported earlier. Since each of the experiments reported have been carried out many times with a variety of oxidase preparations, we are confident that the saturation of a fully reduced oxidase preparation of high purity with CO gas does in fact result in the binding of one CO for each 2 hemes present.

The stoichiometry of 1 CO per 2 heme/copper pairs in Oxi- dase(O1. CO is of interest in relation to the structure of the p- peroxo intermediates (IV], IVIl, I VIII) of Scheme 1,’ as re- cently proposed by Caughey et al. (1). There is a strong experi- mental basis for suspecting the reduction of dioxygen to in- volve formation of an intermediate compound in which perox-

B In this scheme, the fully oxidized oxidase, OxidaseUV), the heme and copper components are envisioned as grouped in two copper/ heme pairs. One pair is loosely coupled magnetically hut is able to exchange electrons readily. This pair, repmsented as C&---F%, is termed the proximal pair because it is only this pair that receives electrons. The electrons enter the oxidase one at a time from cyto- chrome c via Cu, (from the left in Scheme 1). The second pair is tightly coupled magnetically and, presumably, can readily exchange electrons. The distal nair is more remote from the point of electron entry and is represented as F%Xh. Electrons- flow into Oxi- dase(IV) from reduced cymchrome c rapidly m the proximal pair but only slowly to the distal pair. Although with nonphysiological reduc- ing agents such as dithionite, complete reduction to Oxidase(0) is rauidlv achieved. this is not the case with cvtochrome c2+ as reducing agent-In Scheme 1 it is suggested that upon receipt of 1 electron from cvtechrome c*+ (I + II), F%S+ becomes reduced having received an ele&on via Cy; then 0, binda to Fe,,“+ (II -+ III): Thus in classical terms Fe, is the a3 heme.

ide serves as a bridging ligand between two metals (1). Such an intermediate is indicated for heme autooxidations, and the inability to form such a peroxo bridge between hemes due to steric interference by globin has been proposed as one signifi- cant reason for the resistance of HbOp and MbO, to oxidation (34). Although the convincing experimental evidence was ob- tained much later (34), in 1948 Michaelis (35) suggested the inability of 0, to interact with 2 hemes as a possible explana- tion for HbO, stability and in 1957 Wald and Allen (36) sug- gested the interaction of 0, with 2 hemes as a possible reason

CII

1111

[III1

CIVI

[VI

[VII

CVIII

rvrr11

. . .+ Cu2+--Fe3_+"H P P

HOFeit-CCu:+

1 +e. -OH-

. ..+ Cuzt-- Fe2+ 3t 2t P -P

HOFed-Cud

1 +02

. . .+ Cu2+---Fe22+0 P

p 1. HOFe;+-Cu;+

1 +e

. ..+ Cu'+---&,) 2+ P P

w. HOFed3+-Cu d

1 -OH-

. ..+ Cu2t---Fe3~O~0H Fe3+-Cu2+ P P d d

1 +e

-..+ CU'*-- 2+ P -

Fe~~O,OMFe~-CCud

1 +e (Cyt c2+)

Cyt c2+...+ Cu~---Fe~~O.O, Fei+-Cui+

1 +2H+

Cyt t3+. . .* Cu2+---Fe3+OH 3t 2t P P

HOFed- Cu d

1

-Cyt c3+

[II SCHEME 1. Reaction mechanism for cytochrome c oxidase.

CIVal . ..+ C”‘+-&o P P%

HO Cu2fFe3+ cl d

1 -OH-

- CU~+---F~~~~\~,CU~~ Fe:+

[VIa] ...

b e

SCHEME 2. Reaction mechanism for cytochrome c oxidase, altema- tive scheme for Steps IV, V, and VI.

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Infrared Studies of CO Binding to Cytochrome c Oxidase and Hb 5505

for the rapid reduction of 0, by the oxidase. If a peroxo bridge is present, which metals are involved? One metal is surely Fe, because CO binds there and 0, can be expected to do likewise. It is more difficult to decide if the other is Fe, or CQ. If one CO could be shown to bind to each heme iron under certain conditions, this would constitute evidence of each heme iron being available for binding at least under certain conditions. However, since only 1 CO does bind per fully reduced oxidase molecule, either Fe, or Cu, could serve as the second metal to give FG+~~F~,,+~ or Fe,,+zau,fz (V or Va of Schemes 1 and 2 respectively); present evidence does not seem compelling in favor of either one. The inability to demonstrate the binding of CO to each of two metals need not be considered to constitute evidence against peroxide bridge formation since the binding of 1 CO to Fe, could block sterically the access of a 2nd CO to Fe,, or Cu,,.

There is experimental evidence that CO or 0, will bind to either Oxidase(II1) or Oxidase(I1) (37, 38) as well as to more completely reduced species. Indeed there appears no need to involve Oxidase(1) or Oxidase(0) in normal enzyme function (1). Thus, it has been of interest to explore the infrared spectra of CO bound to partially reduced oxidase. Initial experiments suggest that Oxidase(II1). CO and Oxidase(I1). CO exhibit a slightly shifted frequency (vco, 1965 cm-‘); thus, the oxidation state of other components (Cu,, Fe,,, CuJ has a significant, if small, effect upon the bound CO.

Affinities of Hemoglobin and Cytochrome c Oxidase for CO (and 02)-The affinity of Hb for CO is sufficiently strong (as noted below, the K,, is about 4.5 nM) that the complexing is essentially quantitative for dissolved CO free in solution as well as for CO bound to the oxidase. Also, the binding of CO to the oxidase (K,, = 300 nM), although over 50-fold weaker than for Hb, was nevertheless sufficiently strong to permit the Hb to complex essentially all the unbound CO in solution prior to removal of CO from the oxidase carbonyl. The origin of the greater afllnity of Hb for CO can, at least in part, be ascribed to the differences in porphyrin structures for heme A and heme B (Figs. 9 and 10) (2). Particularly important is the strongly electron-withdrawing 8-formyl group of heme A com- pared with the 8-methyl found in heme B. With a common ligand trans to CO such as the histidine imidazole, the Fe(I1) of heme A will be a much less effective n donor to CO (or 021 than will heme B which, in turn, will result in weaker Fe(I1) bonding with CO and in a higher CO stretch frequency (6, 39, 401. Such a trend in frequency versus 2,4substituent on the heme has been observed in reconstituted hemoglobins and

Ho\& d H H2 H H2 H 3

7H2 y 7H2

7” y2

&OH O”c‘OH

FIG. 9. Heme A.

myoglobins (41). That histidine does in fact serve as the ligand trans to CO in Oxidase(0). CO as well as in HbA . CO is of course not firmly established but our infrared data is not inconsistent with the presence of histidine. It is to be noted that infrared spectroscopy provides a direct method for the assessment of the nature of ligand to metal bonding as well as for the determination of the relative affinities of two proteins for a common ligand (or of different ligands for a single pro- t&l).

The differences found among hemeprotein structures do not affect the binding of different ligands to the same degree. For example, Hb A has about 260-fold greater aIEnity for CO than for 0, but with Mbs this difference in affinity is only about 30- to 50-fold. Substitution of only 1 amino acid residue can be important. Thus, in the abnormal human hemoglobin Zurich with the distal histidines of Hb A p chains replaced with arginines, the replacement of CO by 0, is much more difficult in the abnormal than the normal p chains (42). The determi- nation of the relative affinities of CO and 0, for the oxidase is far more difficult to establish than with Hbs and Mbs due to the oxygen reduction reaction of the oxidase. Generally the evidence available suggests the affinity of the oxidase for 0, to be comparable, or even greater, than that for CO. K,,, values for O2 measured for rat liver (43, 44) and brain (451 ranged from 0.02 to 0.5 pM and that for isolated heart oxidase (461 was 0.95 PM. These K,, values may be compared with dissociation constant values of CO for isolated oxidase of 0.3 pM (deter- mined here), 0.6 pM by Wharton and Gibson (271, and 40 pM

by Wainio and Greenlees (47). Pressures of CO somewhat greater than 0, pressures are required to reduce rates by 50% (48,491. From these data it may be concluded that the oxidase has a few fold greater affinity for O1 than does Hb.

That the oxidase should have a greater affinity for 0, than does Hb is, of course, reasonable in terms of their respective physiological roles. Furthermore, it appears advantageous for the oxidase to have CO a less effective competitor of O2 binding than is found with Hb. The efficient operation of the oxidase in energy production requires minimum interference from CO; the oxidase, as an enzyme, “should” have high specificity for its substrate. On the other hand, the role of Hb is to reversibly bind 0, and also to serve in the transfer of small amounts of CO from areas of heme catabolism, where CO is normally generated in small but significant amounts, to the lungs. Thus it is not only important for Hb to transport 0, efficiently to the oxidase but also for it to remove CO at low concentrations from tissue where it can interfere with 0, reduction.

hi2 @ LH I 2 ci

H2

O&lH &OH

FIG. 10. Heme B (protoheme).

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5506 Infrared Studies of CO Binding to Cytochrome c Oxidase and Hb

The structural basis for the difference in relative affinity for CO and 0, found between the oxidase and Hb can be attrib- uted to the effects of protein and porphyrin structure upon the reactivity of iron(B) and to differences in the environment about the bound ligand. The bonding between Fe(II1 and either CO or 0, can be considered “synergistic” in the sense that the low spin Fe(I1) serves as both donor (a rr donor) and acceptor (a acceptor) whereas the ligand is 71 acceptor and u donor (40, 50). O2 appears to withdraw electron density from Fe(II) more effectively than does CO, with NO intermediate in this effect (411.. The ability of heme iron to serve as r donor is an important determinant of the strength of CO bonding as noted from changes in porphyrin or trans.ligand basicity, or both. However, it can be expected that Of, with a much greater affinity for electron density than CO, will bind to the heme iron with much less sensitivity to the subtle differences in electronegativity of the iron which can result from changes in porphyrin structure, in trans-ligand to iron bonding, and in porphyrin-protein interactions. This is not to say that the extent of electron transfer onto Or is such that the bound dioxygen should be regarded as superoxide ligand with ionic iron(lI1) to 02- bonding; rather, a high degree of covalent character can be ascribed to the metal-oxygen atom bond, with the dioxygen moiety more appropriately described as “oxygenyl” in analogy to the terms carbonyl and nitrosyl. The Cro stretch frequency for HbO, (Q = 1106 cm-‘) is within the range where ionic superoxides (e.g. NaO,, K0.J are found (50) but may also result from covalent interactions between Fe(II) and O2 as in carbonyl and nitrosyl bonding where strong covalent bonds with appreciable rr character develop between metal and ligand (50). Such interactions are distinctly differ- ent from ionic bonding between an iron(III1 cation and 02- anion (i.e. superoxide). Only a partial negative charge is expected on the dioxygen ligand from such interactions. This results from a small net transfer of electron density from Fe(H) to O2 when the n donation by Fe(B) is greater than is the o donation by 0,. It should be noted that a comparable positive charge need not develop on iron since the iron is also bound to porphyrin and histidine nitrogens from which it can receive electron density. That the porphyrin does in fact donate such electron density is reflected in the electronic spectra of HbO, and MbOp (41). It is the reactions of bound dioxygen in HbO, or MbOz that provide striking evidence against an anion (super- oxide) being present (50, 51). 0, gas, not O2 , tends to disso- ciate off the protein. To generate 02- from HbO, or MbO, requires a proton-assisted nucleophilic displacement reaction, a very slow process (51). All other known anion replacement reactions (e.g. NS- for Fe, N,- for OH-, etc.1 are very fast, several orders of magnitude faster than are the O,- generating reactions (50, 51). And superoxide, if present, should not be particularly unique as an anion. Thus, the bound 0, reacts precisely as expected for an oxygenyl ligand and does not exhibit reactivities characteristic of an anionic ligand. For these reasons, we consider the term superoxide, used to denote a simple anion bound electrostatically to a ferric cation, to be inappropriate for the O2 of HbO, in view of its reactions and properties. However, the term oxygenyl correctly denotes an analogy with carbonyl and nitrosyl.

The CO and NO stretch frequencies are much more sensi- tive to changes in porphyrin structure in reconstituted hemo- globin than is the M stretch (41, 521, and the shifts in frequency from the gas to ligand for the three gases reveal a greater reduction in bond order for O2 than for CO or NO (50).

Hemoglobin A in which heme B is replaced with 2,4-diacetyl- deuteroheme, a heme with ligand binding properties very similar to heme A, exhibited vo2 at 1106 cm-‘, the same value as HbA02, whereas vco and v&,, values differed by 6 and 4 cm-‘, respectively (41). It may be expected that the differences in prophyrin structure per se between oxidase and Hb will lead to a lower CO affinity and a little changed, or slightly greater, O2 affinity for the oxidase.

The environment about the bound ligand can be expected to influence ligand binding through specific interactions between ligand and protein amino acid residues as well as between ligand and any external medium which may be present. The band widths of ligand infrared bands provide unique informa- tion about such interactions. Ligands bound to hemeproteins generally exhibit very narrow infrared bands if the proteins are in the native conformation (6, 53). Half-band widths for CO bound to Hb and to the oxidase are 8 and 6 cm-‘, respec- tively. Since Hb consists of OL and p subunits with vco values that differ by about 1 cm-’ (7), the half-band widths of the individual subunits are about 7 cm-‘, only slightly broader than is the oxidase CO band. In the absence of protein, heme carbonyls exhibited half-band widths in the range of 12 to 33 cm-’ with a marked dependence upon the nature of the solvent (15). The CO stretch band for both HbCO and MbCO upon lowering the pH was broadened to a half-band width of about 20 cm-’ and the frequency was shifted to 1966 cm-’ (Fig. 11). These changes upon acidification appear due to an unfolding of the native conformations of the globins to give a less struc- tured environment about the bound CO ligand. The carbonyls of HbCO (vco at 1951 cm-‘) and MbCO (vco at 1944 cm-‘, 1933 cm-‘), although in definitely different environments in the native proteins, appeared to be in similar environments upon partial denaturation (unfolding with acid).

In infrared spectroscopy, the band width is determined by the nature of the population of vibrators of different energy that is seen by the incident infrared radiation. If the vibrating dipole interacts with its environment to give a wide range of energies, the band is broad reflecting the energy distribution. On the other hand, a very narrow band is indicative either of little effect of the medium on the vibrating dipole or of a highly ordered environment in which the effects of the medium on the dipole are similar for the entire ligand population. Experi- ments with different solvents reveal that solvation effects differ with the polarity and the shape of the solvent molecules.

EFFECT OF UNFOLDING ON HbCO

0.05M Cbrrate pti 3.0

I V I I, I I 2000 I9 00-2000 l9Of

FREQUENCYbi’)

FIG. 11. Infrared difference sue&a of HbCO minus HbO,: 0.025- nun CaF, cells. Left, BbCO (-lO*mM, 0.1 M potassium phospl&e, pH 7) minus HbO,; rigfit, anaerobically denatured HbCO (-10 mM, 0.05 sodium citrate, pH 3) minus HbO,.

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Infrared Studies of CO Binding to Cytochrom c Oxidase and Hb

Dipole-dipole interactions between solvent molecules and the infrared vibrator affect both the frequency and the intensity of the vibrating dipole seen in the infrared spectrum. The very narrow width for the carbonyl of the oxidase thus means a very narrow range of C-O stretch energies which, in turn, requires a highly uniform polar environment of the CO ligand. The width seen clearly precludes the possibility that the CO is located in the oxidase so as to be surrounded by the aqueous medium external to the protein; in such a case the width would be at least twice as great. The narrow band width found could result from the-complete absence of polar groups in the imme- diate environs of the CO. (With nonpolar groups, their posi- tions with respect to the vibrating dipole of interest will have little effect on infrared energies.) If the CO does, in fact, experience interaction with an adjacent dipole(s), then the relative position(s) of ligand and the polar group(s) must be remarkably uniform; i.e. within the population of CO ligands there can be little variability in the steric positioning of CO with respect to the surrounding groups.

A steric limitation of the preferred linearity in Fd--O bonding is also a possible consequence of the immediate envi- ronment about the CO ligand. Bent Fda bonds were predicted for HbCO and MbCO from an examination of CO stretch frequencies with a greater bending for the Mb than the Hb (4). This conclusion has now been confirmed by neutron (54) and x-ray (55) diffraction studies. Caughey (40) pointed out that greater bending in case of MbCO may be the cause for the lower affinity of CO relative to OZ compared with Hb. Since O2 prefers a bent configuration (Fd ) (50, 56), the

‘0 steric restrictions that affect CO bonding need not similarly destabilize 0, bonding. Analogous steric restrictions on linear F&-O bonding may also occur in the oxidase carbonyl with the resulting destabilization contributing to a lower CO affinity compared with that for Or,

The environment presented by the active site will influence many, if not all, the reactions of the oxidase. The CO ligand infrared data indicates a site that is well isolated from the external medium and, as discussed above, is presumably largely hydrophobic and nonpolar. The nonpolar character of the site will affect the reactions of Scheme 1, indeed those of any mechanistic scheme that might be proposed. Because the overall reaction involves O2 t 4H+ + 4 electrons to give 2 water molecules, changes in charge and counterions must occur. Obviously oxidation-reduction potentials and rates of ionic reactions will differ in a nonpolar hydrophobic medium from what would be the case in an aqueous environment. The four metal centers can change oxidation state and charge, although, according to Scheme 1, only the proximal pair need change oxidation state in the normal reaction. An exchange of charged ligands can be expected but the ligands actually asso- ciated with the coppers or, other than porphyrin, with the irons are unknown. A nonpolar environment will more effec- tively promote the formation of [V] from [IVI than would an aqueous environment. Indeed, an aqueous environment could well stabilize an undesired terminal (i.e. nonbridged) peroxo ligand over the p-peroxo structure of IV1 or [Va]. Also, the reaction required for the splitting of the w bond (VII + VII) discussed earlier (1) would also be particularly sensitive to the polarity of the environment. It will be of great interest to clarify the ligands present at various stages of the oxidase

reactions and to evaluate in greater detail the effects of the environment upon these reactions.

Acknowledgment-We wish to thank Monfort of Colorado, Inc. for the generous gifts of bovine hearts. We wish to ac- knowledge the generosity of Professors David Wharton and Quentin Gibson in providing information to us in advance of publication.

REFERENCES

1.

2.

3. 4. 5.

6. 7.

8.

9.

10. 11.

12.

13.

14.

15.

16.

17

18

19.

20.

21.

22.

23.

24.

25. 26. 27.

28.

29. 30. 31.

32.

33.

Caughey, W. S., Wallace, W. J., Volpe, J. A., and Yoshikawa, S. (1976) in The Enzymes (Bayer, P. D., ed) Vol. 13, pp. 299-344, Academic Press, Inc., New York

Caughey, W. S., Smythe, G. A., O’Keeffe, D. H., Maskasky, J. E., and Smith, M. L. (1975) J. Biol. Chem. 250, 7602-7622

Malmstrom, B. G. (1973) Q. Reu. Biophys. 6, 389-431 Warburg, O., and Negelein, E. (1929) Biochem. 2. 214, 64-106 Keilen, D., and Hartree, E. F. (1939) Proc. R. Sot. Land. Biol.

Sci. 127, 167-191 Alben, J. O., and Caughey, W. S. (1968)Biochemistry 7,175-183 Caunhev. W. S.. Alben. J. 0.. McCov. S.. Charache. S., Hatha-

war, P., and Boyer, S. (1969) Biochemistry 8, 59-62 Cauehev. W. S., Eberspaecher, H., Fuchsman, W. H., McCoy,

ST and Alben, J. 0. (1969) Ann. N. Y. Acad. Sci. 153, 722-737 Caughey, W. S., Bayne, R. A., and McCoy, S. (1970) J. Chem.

Sm. (II). 950-951 -. _- ,, Caughey, W. S. (1971) Adu. Chem. Ser. 100, 248-270 Volpe, J. A., O’Toole, M. C., and Caughey, W. S. (1975) Bio-

them. Biophys. Res. Commun. 62, 48-53 Alben. J. 0.. Yen, L., and Farrier, N. J. (1970) J. Am. Chem.

Sot.’ 92, 4475-4476 Yen, L. (1971) Ph.D. dissertation, Ohio State University,

Columbus, Ohio Maxwell, J. C., Barlow, C. H., Spallholz, J. E., and Caughey, W.

S. (1974) Biochem. Biophys. Res. Commun. 61, 230-236 Maxwell, J. C., and Caughey, W. S. (1976) Biochemistry 15,388-

396 Volpe, J. A., and Caughey, W. S. (1974) Biochem. Biophys. Res.

Commun. 61, 502-509 Choc, M. G., Yoshikawa, S., and Caughey, W. S. (1977) Fed.

Proc. 36, 900 Smith, L. (1955) in Methods ofBiochemical Analysis (Glick, D.,

ed) pp. 427-434, Interscience Publishers, Inc., New York Doeg. K. A., and Ziegler, D. M. (1962) Arch. Biochem. Biophys.

97, 37-40 VanDeBogart, M. E., and Beinert, H. E. (1967) Anal. Biochem.

20, 325-334 Wharton, D. C., and Rader, M. (1970) Anal. Biochem. 33, 226-

229 Geraci, G., Parkhurst, L. J., and Gibson, Q. H. (1969) J. Biol.

Chem. 244, 4664-4667 Banerjee, R., Alpert, Y., Letorrier, F., and Williams, R. J. P.

(1969) Biochemistr 8, 2862-2867 Okunuki, K., Sekuzu, I., Yonetani, T., and Takemori, S. (1958)

J. Biochem. (Tokyo) 45, 847-853 Smith, L. (1955) J. Biol. Chem. 215, 833-846 Cooperstein, S. J. (1963) J. Biol. Chem. 238, 3750-3756 Wharton, D. C., and Gibson, Q. H. (1976) J. Biol. Chem. 251,

2861-2862 Barlow, C. H., Maxwell, J. C., Wallace, W. J., and Caughey, W.

S. (1973) Biochem. Biophrs. Res. Commun. 55, 91-95 Vanneste, W. H. (1966) &chemistry 5, 838-848 Morrison, M., and Horie, S. (1965) J. Biol. Chem. 240.1359-1364 Sharma, V. S., Ranney, H. M., Geibel, J. F., and Traylor, T. G.

(1975) Biochem. Biophys. Res. Commun. 66, 1301-1306 Gibson. 8. H.. Palmer, G., and Wharton, D. C. (1965) J. Biol.

Chem. %IO. 415-920 Maxwell, J. C., and Caughey, W. S. (1977) Method Enzymol., in

press 34. Cohen, I. A., and Caughey, W. S. (1968) Biochemistry 7.636-641 35. Michaelis. L. (1948) Fed. Proc. 7, 509-514

by guest on October 23, 2017

http://ww

w.jbc.org/

Dow

nloaded from

5508 Infiared Studies of CO Binding to Cytochrorne c Oxidase and Hb

36. Wald, G., and Allen, D. W. (195715. Gen. Physiol. 40,593-608 37. Greenwood, C., Wilson, M. T., and Brunori, M. (1974)Biochem.

J. 137, 205-215 38. Anderson, J. L., Kuwana, T., and Hartsell, C. R. (1976) Bio-

chemistry 15, 3847-3855 39. Caughey, W. S. (1967) Annu. Rev. B&hem. 36, 611-644 40. Caughey, W. S. (1970) Ann. N. Y. Acad. Sci. 174, 148-153 41. Caughey, W. S., Maxwell, J. C., Thomas, J. M. G’Keeffe, D. H.,

and Wallace. W. J. (1977) in Metal-Ligund Interactions in Organic Chemistry and Biochemistry (P&man, B., and Gold- blum. N.. ed) Part 2, nn. 131-152, D Reidel Publishing Com- pany; Dordrecht, Ho&d

42. Wallace, W,.J., Volpe, J. A., Maxwell, J. C., Caughey, W. S., and Charache, S. (1976) B&hem. Biophys. Res. Commun. 68. 1379-1386

43. Chance, B. (1964) in Oxygen in Animal Organisms, I. U. B. Symposium No. 31 (Dickens, F., and Neil, E., ed) p. 357, Pergamon Press, Oxford

44. Degn, A., and Wohlrab, H. (1971) Biochim. Biophys. Acta 245, 347-355

45. Clark, J. B., Nicklas, W. J., and Degn, H. (1976)J. Neurochem.

46.

47.

48.

49. 50.

51.

52.

53. 54.

55.

56.

26, 409-411 Petersen. L. C.. Nicholls, P., and Dean H. (1976) Biochim.

Biophyk Acta ‘452, 59-65 - Wainio, W. W., and Greenlees, J. (1960) Arch. Btichem. Bio-

phys. 90, 18-21 Ball, E. G., Strittmatter, C. F., and Cooper, 0. (1951) J. Biol.

Chem. 193. 635-647 Wald, G., and Allen, D. W. (1957)5. Gen. Physiol. 40.593-608 Cauahey, W. S., Barlow, C. H., Maxwell, J. C., Volpe, J. A., and

W&&e, W. J. (1975) Ann. fi. Y. Acud. Sci. 244; l-9 Wallace, W. J., Maxwell, J. C., and Caughey, W. S. (1974)

B&hem. Biophys. Res. Commun. 57, 1104-1110 Maxwell, J. C., and Caughey, W. S. (1974) B&hem. Biophys.

Res. Commun. 60, 1309-1314 McCoy, S., and Caughey, W. S. (1970) Biochemistry 9.2367-2393 Norvell, J. C., Nunes, A. C., and Schoenborn, B. P. (1975)

Science 190, 568-570 Heidner, E. J., Ladner, R. C., and Perutz, M. F. (1976) J. Mol.

Biol. 104, 707-722 Pauling, L. (1949) in Hemoglobin (Roughton, F. W. J., and

Kendrew, J. C., eds) pp. 57-65, Butterworths, London

by guest on October 23, 2017

http://ww

w.jbc.org/

Dow

nloaded from

S Yoshikawa, M G Choc, M C O'Toole and W S CaugheyImplications re O2 reactions.

An infrared study of CO binding to heart cytochrome c oxidase and hemoglobin A.

1977, 252:5498-5508.J. Biol. Chem. 

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