Identification of the Iron-Sulfur Center of Spinach Ferredoxin-Nitrite Reductase as a Tetranuclear Center, and Preliminary EPR Studies of Mechanism*

(Received for publication, August 3, 1978)

Jack R. Lancaster, Jo& M. Vega,+ and Henry Kamin

From the Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710

Nanette R. Orme-Johnson and William H. Orme-Johnson

From the Department of Biochemistry and Institute for Enzyme Research, University of Wisconsin, Madison, Wisconsin 53706

Rick J. Krueger and Lewis M. Siegel

From the Veterans Administration Hospital and the Departments of Botany and Biochemistry, Duke University, Durham, North Carolina 27705

EPR spectroscopic and chemical analyses of spinach nitrite reductase show that the enzyme contains one reducible iron-sulfur center, and one site for binding either cyanide or nitrite, per siroheme. The heme is nearly all in the high spin ferric state in the enzyme as isolated. The extinction coefficient of the enzyme has been revised to Esse = 7.6 X lo4 cm-’ (M heme)-‘. The iron-sulfur center is reduced with difficulty by agents such as reduced methyl viologen (equilibrated with 1 atm of Hz at pH 7.7 in the presence of hydrogenase) or dithionite. Complexation of the enzyme with CO (a known ligand for nitrite reductase heme) markedly increases the reducibility of the iron-sulfur center. New chemical analyses and reinterpretation of previous data show that the enzyme contains 6 mol of iron and 4 mol of acid-labile S2-/mol of siroheme. The EPR spec- trum of reduced nitrite reductase in 80% dimethyl sulf- oxide establishes clearly that the enzyme contains a tetranuclear iron-sulfur (Fe&$*) center. The ferriheme and Fe&* centers are reduced at similar rates (k = 3 to 4 s-‘) by dithionite. The dithionite-reduced Fe&* center is rapidly (k = 100 s-‘) reoxidized by nitrite. These results indicate a role for the Fe4S4* center in catalysis.

The assimilation of nitrate by higher plants and some microorganisms occurs in two steps, the 2-electron reduction of nitrate to nitrite and the 6-electron reduction of nitrite to ammonia. The enzyme responsible for this second step, fer- redoxin-nitrite reductase, has been purified from higher plants by several workers (l-6), and shown to consist of a single polypeptide of molecular weight 61,000 (6). The spinach (7) and Neurospora (8) nitrite reductases were shown to contain the novel prosthetic group siroheme, dimethylurotetrahydro- porphyrin, the complete structure of which has recently been

* This work was supported in part by National Institutes of Health Grants GM-21226 (to H. K.), GM-17170 (to W. O.-J.), and AM-13460 (to L. M. S.) and by Veterans Administration Project 7875-01. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Present address, Department0 de Bioquimica, Facultad de Cien- cias, y CSIC, Universidad de Sevilla, Sevilla, Spain.

elucidated by Scott et al. (9). Siroheme has also been identi- fied as a prosthetic group of Escherichia coli sulfite reductase (10, II), an enzyme which catalyzes the NADPH-dependent reductions of sulfite to sulfide and nitrite to ammonia (12). Both of these reactions involve B-electron reductions. Sulfite reductase has recently been reported to contain an Fe4S4*l center of unusually negative Eo’ (13). Heme ligands such as CO and CN- were found (13) to modify both the reducibility and the EPR signal line shape of the Fe4S4* center, a result interpreted to indicate interaction between the heme and Fe&* centers of the active site.

In 1975, Aparicio et al. (14) described the appearance of an EPR signal characteristic of a reduced iron-sulfur center when cyanide was added to spinach nitrite reductase under reducing conditions. Since then, the existence of enzyme-associated acid-labile S2- and non-heme iron in the enzyme have been verified (6), but quantitation to discriminate between an Fe&?&* or an Fe4S4* center has proven difficult. In 1977, Vega and Kamin (6), working with a preparation of spinach nitrite reductase shown to be homogeneous by the criteria of analyt- ical ultracentrifugation, SDS-polyacrylamide gel electropho- resis, and immunoelectrophoresis, reported analyses indicat- ing 0.6 mol of siroheme, 3.0 mol of iron, and 2.0 mol of acid- labile S”-/61,000 g dry weight of protein. They concluded that the enzyme is composed of one siroheme and one Fe&&* center per peptide chain.

We report here analytical and EPR results that clearly show the presence of an Fe&* center in spinach nitrite reductase. As with the siroheme-Fed&* enzyme sulfite reduc- tase (13), the reducibility of the spinach nitrite reductase Fe&* center is sensitive to the presence of heme ligands. Rapid kinetic studies indicate that the Fe4S4* center reacts quickly enough with reductants and nitrite to play a significant role in the catalytic mechanism of nitrite reduction.

EXPERIMENTAL PROCEDURES

Preparation of Nitrite Reductase-Spinach nitrite reductase was purified as described by Vega and Kamin (6), except that the second ammonium sulfate fractionation was changed from 45 to 55% to 55 to 67% of saturation (15). The purified enzyme exhibited an optical spectrum identical to that reported by Vega and Kamin (6), with the ratio A2&A3R6 less than 2.0, and a specific activity greater than 90

’ The abbreviations used are: Fe&*, tetranuclear iron-sulfur cen- ter; Fe&*, binuclear iron-sulfur center; MV, methyl viologen.

1268

by guest on June 9, 2018http://w

ww

.jbc.org/D

ownloaded from

Fe4-S4* Center in Spinach Nitrite Reductase 1269

units/mg. The preparation showed a single band of protein on SDS- polyacrylamide gel electrophoresis.

Dry Weight Determination-A 0.7~ml volume of purified nitrite reductase was dialyzed uersns three successive 2-liter volumes of 2 mM Tris-HCl, pH 8, for 2 days at 4°C. Duplicate volumes of the dialyzed enzyme and the dialysate were added to each of four pre- weighed Teflon cups and dried at 107°C to constant weight. The cups were weighed on a Cahn microbalance and the weight of the residue determined (2.47 and 2.54 mg for the protein; 0.17 and 0.18 mg for the dialysate). By this method, the extinction coefficient of the nitrite reductase preparation was found to be E386 = 4.03 x IO4 cm-’ (M dry weight)-‘, in good agreement with the value 3.97 x lo* cm-’ (M dry weight)-’ reported by Vega and Kamin (6). Protein concentration was also determined for both the dialyzed and undialyzed enzyme by the method of Lowry et al. (16). One milligram dry weight of protein was equivalent to 0.96 mg of Lowry protein.

Determination of Cyanide Bmding-Purified enzyme, 0.4 ml (Asa = 0.54 cm-‘), in 0.1 M potassium phosphate, pH 7.7, was incubated with 0.1 mM K’%N (4.9 Ci/mol from Amershsm/Searle) for 15 min at 23°C. The enzyme sample then was applied to a Sephadex G-25 column (0.8 x 20 cm) and eluted with the same buffer at a flow rate of 0.5 ml/min. Fractions (1.5-ml) were collected, the absorbance of each fraction at 405 nm was determined, and 20-P aliquots of each fraction were counted for radioactivity. Complete separation of free from bound ‘%N- was achieved as judged by the appearance of a single peak of both radioactivity and absorbance at 405 nm (6), which eluted several fractions before free ‘%N-. The two fractions contain- ing the highest absorbance and radioactivity were pooled. A spectrum was recorded and 50-4 aliquots were measured for radioactivity. The specific radioactivity of the K’%N was determined in a manner essentially identical to that described above using purified methe- moglobin as standard. From this value, moles of bound cyanide per A386 were determined for nitrite reductase. In a separate experiment, it was found that no appreciable change in the spectrum of the cyanide complex of the enzyme occurred upon adding cyanide at concentrations greater than 0.1 mM, indicating that the enzyme is saturated with cyanide at this concentration. The spectrum of the enzymeacyanide complex did not appreciably revert to that of free enzyme even after extensive dialysis, showing that the complex is stable under the conditions used in these experiments.

Determination of Nitrite Binding-Eighty microliters of nitrite reductase (A386 = 4.05 cm-‘) in 0.1 M potassium phosphate, pH 7.7, were placed in one chamber of a small equilibrium dialysis cell. Eighty microliters of a solution of 0.25 mM NaNO* in the same buffer were added to the other chamber. A small segment of dialysis tubing separated the two compartments. The apparatus was placed on a slowly rotating turntable and dialysis continued for 2 h at 23°C. Separate experiments without enzyme showed that nitrite equili- brated across the membrane within this time. The concentration of nitrite in the chamber not containing enzyme was determined by the method of Snell and Snell (17), and found to be 97.5 PM. Assuming equality of volume in both chambers, the concentration of bound nitrite in the chamber containing enzyme could be calculated to be 55 PM. From this number, a value for moles of nitrite bound per AzS6 was determined. It was separately determined that no further change in the optical spectrum of the enzyme (6) occurs at concentrations of nitrite greater than 0.10 mM, showing that the enzyme is saturated at this concentration.

EPR Measurements-EPR spectra were recorded on frozen sam- ples at the temperatures indicated with a Varian model E-9 spectrom- eter operating at X band with field modulation of 100 KHz. Temper- ature was maintained by flow of cooled helium gas using an Air Products helium transfer line. The general procedure for preparation of samples, including the use of a vacuum manifold for anaerobic manipulations, was as previously described (18). The preparation of freeze-quenched rapid reaction samples for EPR spectroscopy was performed by the modified method of Bray (19), using a System 1000 Precision Syringe Ram from Update Instruments, Inc.

For quantitation of spins, all spectra were recorded under non- saturating conditions, at the following temperatures: high spin ferri- heme, 9 K; heme.NO complex, 25 K; reduced iron-sulfur center, 13 K. The high spin ferriheme was quantitated by integration of the g = 6.78 peak according to the method of Aasa and Vangaard (20). The hemoprotein subunit of E. coli sulfite reductase served as standard. Studies of the temperature dependence of the magnitude of the integral of the g > 6 features of the two enzymes showed that the

8X” iii

D305 0310 0315 032U a325 Q330 0335 0340 a3

0.30 0.31 0.32 033 t MAGNETIC FIELD, T

FIG. 1. EPR spectra of nitrite reductase. All solutions contained 0.1 M potassium phosphate, pH 7.7. A, oxidized enzyme (75 PM in heme). Conditions of EPR measurement: temperature, 20 K; micro- wave power, 50 milliwatts; frequency, 9.19 GHz; modulation ampli- tude, 1 mT. B, enzyme.NO complex. A solution containing 4 PM (in heme) enzyme, 1 mM methyl viologen, and 1 mM Na2S204 was incu- bated anaerobically under 0.7 atm of NO for 1 h at 25°C. Conditions of EPR measurementi temperature, 13 K; microwave power, 30 mi- crowatts; frequency, 9.20 GHz; modulation amplitude, 1 mT. C, iron- sulfur center in reduced enzyme.CO complex. Nitrite reductase, 35 PM (in heme), was incubated for 2 h at 23°C in the presence of 24 pM spinach ferredoxin, 0.025 volumes of a ferredoxin-free extract of C. pasteurianum which contained the pyruvate dehydrogenase as described by Lovenberg et al. (23), 50 pM CoA, and 10 mM pyruvate, under 1 atm of CO. A control solution, incubated in parallel, contained all of the components except nitrite reductase. The difference EPR spectrum between the two solutions, obtained with the aid of a digital computer, is shown. Conditions of EPR measurement; temperature, 13 K; frequency, 9.19 GHz; microwave power, 0.3 milliwatt; modula- tion amplitude, 1 mT.

corrections due to incomplete population of the lowest Kramers doublet at 9 K were negligible (21). The spectra of the heme.NO complex and reduced iron-sulfur center were quantitated by double integration, with a solution of cupric EDTA as standard.

Optical Spectra-Absorption spectra were recorded on a Varian 219 or American Instrument DW-2 spectrophotometer at 23°C in silica cells of l-cm path length.

Chemical Analyses-Analysis of nitrite reductase for total iron and acid-labile S*- were performed as described by Vega and Kamin (6). Siroheme was measured in the acetone/HCl extract of nitrite reductase to which pyridine had been added, according to the proce- dure of Siegel et al. (22).

RESULTS AND DISCUSSION

Heme Content-An EPR spectrum of spinach nitrite reduc-

tase is shown in Fig. 1A. The oxidized enzyme spectrum is

that of a high spin ferriheme species with g values at 6.78,

by guest on June 9, 2018http://w

ww

.jbc.org/D

ownloaded from

1270 Fed-S4* Center in Spinach Nitrite Reductase

TABLE I

Analysis of nitrite reductase for heme, iron-sulfur centers, and ligand binding sites

For detaiIs of analytical methods and EPR quantitation see “Ex- perimental Procedures” and the text. All chemical and EPR analyses were performed on at least two different enzyme preparations; chem- ical analyses were performed at least in duplicate on each preparation. Results differed in all cases by no more than *lo%.

component Mo1/61,0XI g Mol/mol siro- enzyme heme

Siroheme Chemical analysis High spin ferriheme (EPR) Ferroheme-NO (EPR)

Iron-sulfur centers

0.52 1.00 0.48 0.92 0.49 0.94

Total iron (chemical analysis) Labile S*- (chemical analysis) Reduced Fe-S center (EPR, reduced

enzyme. CO complex)

3.1 5.9 2.0 3.8 0.46 0.88

Binding sites Cyanide 0.59 1.13 Nitrite 0.61 1.17

5.17, and 1.98. Chemical analyses of the enzyme for siroheme by the pyridine hemochromogen technique (22) showed 0.52 mol of siroheme/g-molecular weight of protein (on a dry weight basis). Quantitation of the high spin ferriheme EPR spectrum yielded 0.48 mol/mol dry weight. Quantitation of the ferroheme.NO complex EPR spectrum (see below) yielded a value of 0.49 mol/mol dry weight (Table I).

Aparicio et al. (14) have reported that incubation of spinach nitrite reductase with nitrite and the reducing agent dithionite results in disappearance of the high spin ferriheme EPR signals and the appearance of a new set of EPR signals with principal g values at 2.07 and 2.00. They attributed this signal to formation of a ferroheme. NO complex. We have confiied this observation and have found, in addition, that the g = 2.07 species is formed upon addition of either hydroxylamine (with or without added reductant), or a reductant (dithionite or reduced methyl viologen) plus NO itself to the enzyme. The EPR spectrum of the nitrite reductase.NO complex, shown in Fig. lB, exhibits hyperfine splitting with a coupling constant of 2.1 mT in agreement with the presence of a nitrogen ligand.’ Integration of the ferroheme . NO spectrum yielded 0.49 mo1/61,000 g of protein. An identical value for quantitation of the ferroheme . NO signal was obtained in an experiment in which 8 PM enzyme (dry weight basis) was reacted for 90 s with 15 mM KNO?, 50 mM Na&O1, and 35 pM methyl viologen. Increased time of reaction resulted in no change in the amount of heme. NO signal obtained. Thus, following turnover with NO1 and the reductants employed in this experiment, sub- stantially all of the enzyme heme is in the form of a ferroheme. NO complex.

Binding Sites for Cyanide and Nitrite-Vega and Kamin (6) showed that oxidized nitrite reductase readily forms spec- trophotometrically detectable complexes with cyanide (Kd = 4.3 PM) or nitrite (Kd = 3.2 PM). The EPR spectrum of the enzyme has been reported to be markedly altered upon addi- tion of either cyanide or nitrite (14, 26) indicating that these agents bind at or close to the enzyme heme. We have deter- mined the number of binding sites in our enzyme preparation for each of these ligands. The data of Table I show that nitrite reductase binds approximately 0.6 mol of either cyanide or nitrite/61,000 g of protein, i.e. just over 1 mol of ligand/mol of siroheme.

* The nearly axial nature of the putative nitrite reductase-NO EPR spectrum is unusual for ferroheme. NO complexes. A similar spectrum has been reported, however, for hemoglobin-NO in the presence of 0.2 M SDS (25).

Reducibility of Iron-Sulfur Center-Stoller et al. (27) and Cammack et al. (26) have reported the presence of an EPR signal attributed to an iron-sulfur center in plant nitrite re- ductases treated with reducing agents. They concluded that the apparent oxidation-reduction potential of this center was unusually negative (less than -550 mV at pH 8).

The data of Table II show that ability to reduce the iron- sulfur center of spinach nitrite reductase depends not only on the nature and reducing strength of the reductant used but can be markedly influenced by addition of a substance, CO, known to bind to the heme moiety of the enzyme (6). Thus, only a small “g = 1.94” type of EPR signal was detected when enzyme was incubated with methyl viologen equilibrated with 1 atm of H2 at pH 7.7 in the presence of the enzyme hy- drogenase. (Such incubation procedures have previously been shown to lead to reduction of a number of bacterial and plant ferredoxins (28).) Addition of CO to this incubation mixture resulted in a lo-fold increase in intensity of the reduced iron- sulfur center EPR signal. Addition of dithionite, a stronger reductant than the HJhydrogenase system at pH 7.7, to the reduced enzyme.CO complex resulted in a further 2.4-fold increase in intensity of the reduced iron-sulfur center EPR signal. The signal elicited by addition of S,O?‘- to enzyme in the absence of CO is considerably smaller than that observed upon S204’- addition to the preformed enzyme. CO complex.’ Cyanide (1 mM), like CO, was found to increase markedly the ability of the nitrite reductase iron-sulfur center to be reduced by the HJhydrogenase/methyl viologen system. Cammack et al. (26) have shown that CO, CN-, and NH:% can cause quali- tative changes in line shape and reducibility of the iron-sulfur center of Cucurbita pepo nitrite reductase.

The EPR spectrum obtained upon incubation of nitrite reductase with CO in the presence of a reduced ferredoxin- generating system is shown in Fig. 1C. The spectrum is nearly axial, with g values at 2.04 and 1.94. Quantitation of this spectrum yielded 0.46 mol of iron-sulfur center/61,000 g of protein, i.e. approximately 0.9 centers/heme.

Fe2S2* or Fe+‘!&* Center?-We have repeated the chemical analyses of Vega and Kamin (6) for total iron and acid-labile S2- with the present enzyme preparations, on which the quantitative data of Table I were obtained. Our analyses confirm the presence of 3.0 + 0.2 mol of iron and 2.0 f 0.2 mol of S’~/61,000 g of protein. When these data are taken together with the other results of Table I, it can be concluded that nitrite reductase contains approximately 6 mol of iron and 4 mol of S2-/heme. The EPR results show that there is one iron-sulfur center/heme. The data thus support the presence of an Fed&* rather than an Fe&* center in nitrite reductase.

Cammack (29) and Cammack and Evans (30) have shown that certain denaturing agents, including guanidine HCl, di- methylformamide, or dimethyl sulfoxide, cause the protein moieties of a number of iron-sulfur proteins to unfold while the iron-sulfur centers remain intact. The EPR spectra of the reduced proteins, unfolded by dimethyl sulfoxide treatment, are characteristic of the type of center present (29,30). Fe&* centers give rise to a rather isotropic signal which is readily detected at temperatures greater than 77 K, but which is easily saturated with microwave power at 20 K. Fe&* centers

’ Spectrophotometric and inhibition studies by Vega and Kamin (6) have shown that addition of dithionite to nitrite reductase prior to or simultaneously with treatment of the enzyme with CO prevents formation of the enzyme. CO complex, presumably due to formation of a competing complex between the enzyme heme and a sulfur compound (possibly sulfite) derived from dithionite. Table II shows that a much smaller reduced iron sulfur center EPR signal is observed when dithionite and CO are added together to the enzyme (Sample 6) than when dithionite is added to the preformed enzyme.CO complex (Sample 3).

by guest on June 9, 2018http://w

ww

.jbc.org/D

ownloaded from

Fe.&&* Center in Spinach Nitrite Reductase 1271

give an axial or near axial signal which is only detected below 35 K, and which is not readily saturated with microwave power at 20 K.

Fig. 2 shows the EPR spectrum of reduced nitrite reductase in the presence of 80% dimethyl sulfoxide. The spectrum, taken at 20 K and 10 milliwatts of power, is nearly axial, with g values of 2.04 and 1.93. No saturation of the signal was detected at 20 K until powers of greater than 25 milliwatts were applied. The spectrum could not be detected at 77 K. The behavior of nitrite reductase in dimethyl sulfoxide thus corresponds to that of an Fe&* center-containing protein.

Rapid Kinetic Studies-Previous spectroscopic and inhi- bition studies have implicated the siroheme prosthetic group in the catalytic mechanism of nitrite reduction (6). Steady state kinetic studies by Cammack et al. (26) have suggested a

TABLE II

Effect of CO on reducibility of nitrite reductase iron-sulfur center All samples contained 4 pM (in heme) nitrite reductase in 0.1 M

potassium phosphate, pH 7.7, and 1 mM methyl viologen (MV). In Samples 1 to 4, the nitrite reductase was equilibrated with 1 atm of Hz and MV at pH 7.7 in the presence of 0.1 pM Clostridium pasteu- rianum hydrogenase (prepared according to Tso et al. (24)). Sample 1 was incubated under 1 atm of H2 for 2 h. Sample 2 was identical to Sample 1, except that after 1 h of incubation under 1 atm of HZ, the atmosphere was replaced with CO and the incubation was continued for an additional 1 h. Sample 3 was identical to Sample 2, except that 1 mM Na2S204 was added after the l-h incubation under CO, and the incubation was continued an additional 5 min. Sample 4 was identical to Sample 1, except that 1 mM Na&04 was added after the 2-h incubation with Hz, and the sample was incubated an additional 5 min. In Sample 5, enzyme was incubated for 2 h with 1 mM methyl viologen and 1 mM Na2S204 under an atmosphere of NP. Sample 6 was identical to Sample 5, except that it was incubated under an atmosphere of CO. Incubations were at 23°C. Samples were frozen in liquid NP and EPR spectra were recorded at 13 K, 3 milliwatts of microwave power, and 1 mT modulation amplitude. Intensity of reduced iron-sulfur center signals was measured by amplitude of the g = 1.94 feature. Hzase, hydrogenase.

Additions of enzyme Relative Fe-S cen- ter signal

1. MV/H*/Hsase 4 2. MV/Ha/Haase, then CO 40 3. MV/H;?/Hnase, then CO, then S204 2- 100 4. MV/Hz/Hzase, then SnOs 2- 21 5. MV/Sz04*- 13 6. MV/&04’-/CO 17

033 034 035 036 037 MAGNETIC FIELD, T

FIG. 2. EPR spectrum of reduced nitrite reductase iron-sulfur cen- ter in 80% dimethyl sulfoxide. Dimethyl sulfoxide, 0.2 ml, was added anaerobically to 0.05 ml of a solution containing 0.37 mM (in heme) nitrite reductase, 10 mM Na&04, and 0.1 M potassium phosphate, pH 7.7. The solution was maintained just above the freezing point of dimethyl sulfoxide with an ice bath, and the dimethyl sulfoxide was added in four equal aliquots, with 15 s of cooling of the solution in an ice bath after each addition. Conditions of measuremenk temperature, 20 K; microwave frequency, 9.14 GHz; microwave power, 10 milliwatts; modulation amplitude, 1 mT. The small radical signal at g = 2.00 was present in a control solution from which enzyme had been omitted.

J

TIME (set)

FIG. 3. Kinetics of reduction of nitrite reductase heme and iron- sulfur center by dithionite. An anaerobic solution of 34 pM (in heme) nitrite reductase in 0.1 M potassium phosphate buffer, pH 7.7, was rapidly mixed with an equal volume of an anaerobic solution contain- ing 0.1 M Na&204 in the same buffer. After reaction at 22°C for the time periods indicated in the figure, the reaction mixtures were sprayed into isopentane maintained at 130 K to stop the reaction, and the samples were analyzed by EPR spectroscopy. The point corresponding to maximal signal amplitude of the iron-sulfur center EPR signal was obtained after 75 s of reaction. The “zero time” point was obtained by rapidly mixing 34 pM enzyme for 6 ms with anaerobic buffer. EPR spectra were recorded at 13 K and 3 milliwatts of microwave power. Ferriheme was measured by means of the ampli- tude of the g = 6.78 EPR signal. Reduced iron-sulfur center was measured by means of the amplitude of the EPR signal at g = 1.94.

OO- TIME (WC)

I 0

FIG. 4. Kinetics of oxidation of the reduced iron-sulfur center of nitrite reductase by nitrite. An anaerobic solution containing 43 PM (in heme) nitrite reductase and 0.1 M Na&04 in 0.1 M potassium phosphate, pH 7.7, was mixed rapidly with an equal volume of an anaerobic solution containing 15 mM NaNOz in the same buffer. After reaction at 22°C for the time period indicated in the figure, reaction mixtures were sprayed into isopentane maintained at 130 K to stop the reaction, and the samples were analyzed by EPR spectroscopy. The “zero time” point was obtained by rapidly mixing the enzyme/dithionite solution for 6 ms with anaerobic buffer. EPR spectra were recorded at 13 K and 3 milliwatts of microwave power. Reduced iron-sulfur center was measured by means of the amplitude of the EPR signal at g = 1.94.

role for both the iron-sulfur center and the ferroheme.NO complex in the reaction mechanism. No rapid kinetic studies on the enzyme have as yet been reported. Fig. 3 shows the time course of reduction by dithionite of the high spin ferri- heme and the iron-sulfur centers of nitrite reductase. Both centers are reduced on a comparable time scale (k = 3 to 4

by guest on June 9, 2018http://w

ww

.jbc.org/D

ownloaded from

1272 Fe4-S4* Center in Spinach Nitrite Reductase

s-l). The relatively slow nature of the reduction may be correlated with the low turnover number at pH 7.7 determined for dithionite-mediated nitrite reduction catalyzed by the enzyme (2 mol of NOam s-l heme-’ compared with 200 mol of NO*- s-l heme-’ when dithionite-reduced methyl viologen serves as reductant). Fig. 4 shows that the iron-sulfur center of enzyme prereduced with dithionite is rapidly reoxidized upon addition of nitrite (h = 100 s-l). This data strongly supports a role for the iron-sulfur center in the mechanism of nitrite reduction.

Nitrite Reductase Extinction Coefficient-Vega and Kamin (6) have reported an extinction coefficient for the enzyme of Ezs6 = 4.0 X lo4 cm-’ (M enzyme dry weight))‘. We have confirmed this finding. The data of Table I show that the enzyme preparations, reported to be homogeneous by a num- ber of criteria of protein chemistry, contain 0.53 ? 0.05 active centers (i.e. prosthetic groups or ligand binding sites) per mol dry weight. Thus, an extinction coefficient of Es86 = 7.6 X lo4 cm-’ (M active center))’ would seem to be more appropriate in considering quantitation of enzyme active centers during studies of the catalytic mechanism. There is a high likelihood that even the purest preparations described contain extra protein, perhaps apoenzyme.4

Knaff et al. (31) have recently reported finding a 1:l com- plex between spinach ferredoxin and nitrite reductase by spectrophotometric titration studies. In interpreting their ex- periments, they used the Ezs6 of 4.0 x lo4 cm-’ M-’ to deter- mine nitrite reductase concentration. If ferredoxin forms a complex with enzyme devoid of prosthetic groups as well as with enzyme containing heme and Fe&* centers, the conclu- sion of a 1:l complex could remain valid. However, if ferre- doxin binds only to enzyme molecules containing heme and Fe.&* centers, the data of Knaff et al. (31) suggests formation of a tight 2:l complex between ferredoxin and nitrite reduc- tase. Such questions can be resolved if enzyme preparations free of inactive protein can be obtained. We are presently attempting to obtain enzyme free of such material.

’ Vega and Kamin (6) based their conclusion of a binuclear iron- sulfur center in part upon heme analysis plus the apparent homoge- neity of the protein. They assumed, from the relatively low analytical values for heme, that their heme extraction had been incomplete. In fact, as shown here, their heme analyses were substantially correct (Table I). Thus, some of the protein of the preparations described here and previously (6) must be devoid of prosthetic groups. An alternate explanation, that the enzyme is a dimer of molecular weight approximately 120,ooO to 130,006 (with prosthetic groups on only one of the subunits) is unlikely since independent physical measurements (6) all indicate a molecular weight of about 61,060, with no evidence for aggregation.

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.

REFERENCES

Cardenas, J., Barea, J. L., Rivas, J., and Moreno, C. G. (1972) FEBS Lett. 23, 131-135

Zumft, W. G. (1972) Biochim. Biophys. Acta 276, 363-375 Shimuzu, J., and Tamura, G. (1974) J. Biochem. (Tokyo) 75,

999-1005 Ida, S., Kobayakawa, K., and Morita, Y. (1976) FEBS Lett 65,

305-308 Hucklesby, D. P., James, D. M., Banwell, M. J., and Hewitt, E.

(1976) Phytochemistry 15,599-603 Vega, J. M., and Kamin, H. (1977) J. Biol. Chem. 252,896-909 Murphv, M. J., Siegel, L. M., Tove, S. It., and Kamin, H. (1974)

PI& Natl. Acnci:Sci. 1J. S. A. 71, 612-616 Veea. J. M.. Garrett. R. H.. and Siegel. L. M. (1975) J. Biol.

i;hkm. 256, 7980-7489 - Scott, A. I., Irwin, A. J., Siegel, L. M., and Schoolery, J. A. (1978)

J. Am. Chem. Sot. 100,316-318 Siegel, L. M., Murphy, M. J., and Kamin, H. (1973) J. Biol. Chem.

248, 251-264 Murphy, M. J., Siegel, L. M., Kamin, H., and Rosenthal, D. (1973)

J. Biol. Chem. 248, 2801-2814 Siegel, L. M., Davis, P. S., and Kamin, H. (1974) J. Biol. Chem.

249, 1572-1586 Siegel, L. M. (1978) in Mechanisms of Oxidizing Enzymes

(Singer, T. P., and Ondarza, R. N., eds) pp. 201-214, Elsevier- North Holland, New York

Aparicio, P. J., Knaff, D. B., and Malkin, R. (1975) Arch. Biochem. Biophys. 169, 102-107

di Jeso, F. (1968) J. Biol. Chem. 243, 2022-2023 Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J.

(1951) J. Biol. Chem. 193,265-275 Snell, F. D., and Snell, C. T. (1949) Calorimetric Methods of

Analysis, p. 804, D. van Nostrand Co., New York Orme-Johnson, W. H., Hamilton, W. I)., Ljones, T., Tso, M.-Y.

W., Burris, H. H., Shah, V. K., and Brill, W. J. (1972) Proc. Natl. Acnd. Sci. I/. S. A. 69, 3142-3145

Bray, K. C. (1961) Biochem. J. 81, 189-193 Aasa, H., and Vangaard, T. (1975) J. MagnetLc Hes. 19, 308-315 Peisach, J., Blumberg, W. E., Ogawa, S., Kachmilewitz, E. A., and

Oltzik, R. (1971) J. Biol. Chem. 246, 3342-3355 Siegel, L. M., Murphy, M. J. and Kamin, H. (1978) Methods

Ehymol. 51, 436-446 Lovenberg, W., Buchanan, B. B., and Habinowitz, J. C. (1963) J.

Bud. Chem. 238, 3899-3913 Tso, M. W., Ljones, I’., and Burris, K. H. (1972) Biochim. Biophys.

Actn 267, 600-604 Kon, H. (1968) ,I. Biol. Chem. 243,4350-4357 Cammack, R., Hucklesby, D. P., and Hewitt, E. J. (1978) Biochem.

J. 171,519-526 Stoller, M. L., Malkin, R., and Knaff, D. B. (1977) FEBSLett. 81,

271-274 Stombaugh, N. A., Sundequist, J. E., Burris, R. H., and Orme-

Johnson, W. H. (1976) BEochemistry 15,2633-2641 Cammack. R. (1975) Biochem. Sot. Trans. 3.482-489 Cammack; R., and Evans, M. C. W. (1975). Biochem. Biophys.

Res. Commun. 67, 544-549 Knaff, D. B., Smith, J. M., and Malkin, R. (1978) FEBS Lett. 90,

195-197

by guest on June 9, 2018http://w

ww

.jbc.org/D

ownloaded from

Krueger and L M SiegelJ R Lancaster, J M Vega, H Kamin, N R Orme-Johnson, W H Orme-Johnson, R J

tetranuclear center, and preliminary EPR studies of mechanism.Identification of the iron-sulfur center of spinach ferredoxin-nitrite reductase as a

1979, 254:1268-1272.J. Biol. Chem. 

  http://www.jbc.org/content/254/4/1268Access the most updated version of this article at

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

to choose from all of JBC's e-mail alertsClick here

  http://www.jbc.org/content/254/4/1268.full.html#ref-list-1

This article cites 0 references, 0 of which can be accessed free at

by guest on June 9, 2018http://w

ww

.jbc.org/D

ownloaded from