coordination chemistry of f430 - journal of biological chemistry

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 264, No. 19, Issue of July 5, pp. 11236-11245,1989 Printed in U. S. A.

Coordination Chemistry of F430 AXIAL LIGATION EQUILIBRIUM BETWEEN SQUARE-PLANAR AND BIS-AQUO SPECIES IN AQUEOUS SOLUTION*

(Received for publication, October 26, 1988)

Andrew K. ShiemkeS, John A. Shelnuttj, and Robert A. ScottSll From $Departments of Chemistry and Biochemistry and Center for Metalloenzyme Studies, University of Georgia, Athens, Georgia 30602 and $Fuel Science Division 6211, Sandia National Laboratories, Albuquerque, New Mexico 87185

X-ray absorption spectroscopic characterization of axial ligand coordination to factor F430, the nickel- tetrapyrrole cofactor of the S-methyl-coenzyme M (CH3SCoM) methyl reductase enzyme from methano- genic bacteria, is presented. The nickel of isolated F430 is hexacoordinate at 10 K in aqueous solution (as is the enzyme-bound cofactor), whereas the epimerized and ring-oxidized derivatives of F430 have four-coordinate nickel. Reduction of the ring-oxidized derivative, F660, with dithionite yields F430 in its native configuration, with axial ligands indistinguishable from those present when the cofactor is obtained directly from the holoenzyme. Thus, we conclude that the axial ligands to F430 in aqueous solution are water molecules. Analysis of the nickel extended x-ray absorption fine structure is consistent with this conclusion. Resonance Raman spectra obtained at room temperature contain features characteristic of both 4- and 6-coordinate forms of the cofactor. We have found that the resonance Raman, optical, and x-ray absorption spectra of aqueous solutions of F430 are temperature-dependent due to a ligand-binding equilibrium involving the square-planar and 6-coordinate bis-aquo forms of the cofactor. At low temperatures (<250 K) the 6-coordinate form pre- dominates, whereas higher temperature solutions contain both 4- and 6-coordinate species in a dynamic equilibrium. Similar behavior is observed in other weakly coordinating solvents such as methanol and ethanol. The 4-coordinate form is predominant in solvents with strong electron-withdrawing substituents such as 2,2,2-trifluoroethanol and 2-mercaptoethanol. The relevance of this facile ligand exchange to the active site structure and enzymatic mechanism of the parent enzyme is discussed.

The methanogens constitute a diverse class of archaebac- teria, many of which are capable of living autotrophically on hydrogen and carbon dioxide (Daniels et al., 1984). These bacteria derive energy from the stepwise reduction of carbon dioxide to methane (the methanogenesis cycle), in which the C1 fragment is passed between a series of unusual cofactors (Wolfe, 1985; Rouvibre and Wolfe, 1988). S-Methyl-coenzyme

* This work was supported by National Science Foundation Pres- idential Young Investigator Award CHE 84-51684/87-15889 (to R. A. S.), and with United States Department of Energy Contract DE- AC04-76DP00789 and Gas Research Institute Contract 5082-260- 0767 (to. J. A. S.). 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.

7l To whom correspondence should be addressed.

M (CH3SCoM)’ methyl reductase is the terminal enzyme in this pathway, catalyzing the reductive cleavage of CH3SCoM to methane and coenzyme M (HSCoM) (Ellefson et al. 1982). Thermodynamic considerations indicate that this terminal reaction is the only energetically favorable reaction in methanogenesis and appears to be coupled somehow to the production of a chemiosmotic membrane potential (Blaut and Gottschalk, 1985). Methyl reductase activity can be reconsti- tuted in vitro with a system containing the enzyme, CH3SCoM, and HSHTP (N-7-mercaptoheptanoyl-0-phospho-L-threonine) (No11 and Wolfe, 1987; Ellerman et al., 1988). HSHTP appears to act as reductant in this system with the result that methane and a mixed disulfide of HSCoM and HSHTP are produced (Bobik et al., 1987; Bobik and Wolfe, 1988; Ellerman et al., 1988) (Scheme 1, reaction 1) :

R-H vm H2

SCHEME 1

Recent results indicate that HSHTP is a degradation product of the physiological form of this cofactor resulting from the loss of two N-acetylglucosamine residues and an additional unidentified group (Sauer et al., 1987). Thauer and coworkers have recently identified the enzyme responsible for the reductive cleavage of the heterodisulfide product of the CH3SCoM reduction (Scheme 1, reaction 2 ) (Hedderich and Thauer, 1988).

Methyl reductase contains a nickel tetrapyrrole known as factor F430 (Ellefson et al., 1982). This cofactor is a yellow, non-fluorescent compound which derives its name from an intense optical absorption band at 430 nm ( e = 23,300 M” cm”) (Hausinger et al., 1984). The structure of the F430

macrocycle is shown in Fig. 1 (Pfaltz et al., 1982; Livingston

The abbreviations used are: CH3SCoM, 2-methylthioethanesul- fonic acid; HSCoM, coenzyme M (2-mercaptoethanesulfonic acid); methyl reductase, CH3SCoM methyl reductase enzyme; HSHTP, N - 7-mercaptoheptanoyl-O-phospho-~-threonine; XAS, x-ray absorption spectroscopy; EXAFS, extended x-ray absorption fine structure; FT, Fourier transform; RR, resonance Raman; HPLC, high pressure liquid chromatography; OEpc, octaethylpyrrocorphin; BM, Bohr magnetons.

11236

Coordination Chemistry of F430 11237

et al., 1984). Also shown in Fig. 1 are structures of the configurational isomers produced by thermal isomerization of F430 (Diekert et al., 1981; Pfaltz et al., 1985). In these isomers the stereochemical orientation of the side chains attached to CI3, or CI2 and CI3 are reversed (epimerized) relative to their configuration in the native cofactor, yielding the 13-monoepimer and the 12,13-diepimer, respectively. Ring C of the F430 macrocycle is also susceptible to oxidation yielding 12,13- didehydro F430 (also known as Fsso), which differs from the cofactor by the presence of a double bond between the p- carbons of this pyrrolidine ring (Pfaltz et al., 1985), as shown in Fig. 1.

The specific role of F430 in the reductive cleavage of CH3SCoM is unknown, but it is thought to be the site of substrate reduction. Possible roles for F430 in this reaction include substrate binding (CH3SCoM and/or HSHTP), electron transfer from HSHTP to CH3SCoM, or methyl-group transfer. Results of whole-cell EPR spectra indicate that at least one Ni(1) form of enzyme-bound F430 participates in the production of methane from hydrogen and carbon dioxide (Albracht et al., 1988). Multiple Ni(1) forms of the enzyme are observed under different conditions, and it was speculated that they may differ with respect to the nature of the axial ligands (Albracht et al., 1988). The study of the axial coordination chemistry of F430 is therefore crucial to the understand- ing of the unusual chemistry of the methyl reductase enzyme.

In previous studies we have presented evidence from x-ray absorption spectroscopy (XAS) that F430 contains pseudo- octahedrally coordinated nickel at 10 K in aqueous solution (Eidsness et al., 1986; Shiemke et al., 1988a). T h e closely related configurational isomers of F430 (the 13-monoepimer and 12,13-diepimer) contain nickel in a square-planar geometry under similar conditions (Shiemke et al., 1988a, 1989). These different coordination geometries are related to differ- ing macrocycle conformations, with native F430 having an expanded core y d presumably planar macrocycle (Ni-N distances of -2.1 A), whereas the configurational isomers have contracted ando presumably ruffled macrocyles (Ni-N distances of -1.9 A (Shiemke et al., 1988a, 1989)). These differences in chromophore structure have been correlated with differences in resonance Raman (RR) and optical spectral properties (Shiemke et ul., 1988a, 1988b, 1989). However, the RR spectrum of F430 in aqueous solution at room temperature contains features of the macrocycle conformations associated with both the 4- and 6-coordinate complexes of F430 (Shiemke et al., 1988b). We have resolved this apparent conflict with the XAS results by demonstrating the existence of a dynamic ligation equilibrium for F430 in aqueous solution near room temperature. Raman spectra at low temperature (77 K) indicate the presence of only the 6-coordinate form, whereas x- ray absorption, RR, and optical spectra obtained at room temperature contain features of both 4- and 6-coordinate species. Additional spectroscopic evidence is presented that indicates the axial ligands involved in this equilibrium are supplied by the solvent.

EXPERIMENTAL PROCEDURES

Materials-Chromatographic media (phenyl-Sepharose, Q-Sepha- rose, QAE-Sephadex A-25, and DEAE-Sephadex A-25) were pur- chased from Pharmacia LKB Biotechnology Inc. Chemicals and solvents were reagent grade or better and the solvents (methanol, ethanol, 2,2,2-trifluoroethanol, and 2-mercaptoethanol) were vac- uum-distilled prior to use.

Methanobacterium therrnoautotrophicum (strain A H ) cells were the kind gift of Dr. R. S. Wolfe (University of Illinois). The cells were grown on hydrogen and carbon dioxide as energy and carbon sources. The cells were grown at 65 “C, harvested, and stored under nitrogen at -20 ‘C as described previously (Gunsalus et aL, 1978).

Protein and Cofactor Purification-Methyl reductase was purified from the soluble fraction after disruption of the cells in a French press at 16,000 p.s.i. and centrifugation at 24,000 X g for 90 min. After a preliminary ammonium sulfate precipitation to remove most of the contaminating proteins, the methyl reductase was purified by chromatography on phenyl-Sepharose and fast-flow Q-Sepharose, according to published procedures (Shiemke et al., 1988a). F430 was extracted from the holoenzyme by suspending the protein in a 2 N solution of LiCl or LiBr in 80% aqueous ethanol, as described previously (Shiemke et al., 1988a). The 13-monoepimer, the 12,13-diepimer, and F560 were purified from the pool of “free” F430 by chromatography on DEAE-Sephadex A-25 and QAE-Sephadex A-25 (Shiemke et al., 1988a). In the final purification step of free F430 with 1.5 X 60- cm QAE-Sephadex column and a flow rate of 30 ml/h, the native isomer eluted between 1.1 and 1.3 liters and was followed by the 12,13-diepimer at 1.3-1.6 liters, the 13-monoepimer at 1.8-1.95 liters, and F, a t 1.95-2.05 liters. Ammonium formate was removed by multiple steps of lyophilization and dissolution with 18 megaohm-cm water. Base-line separation of these four species could be routinely obtained in this manner, provided the total sample load is not excessive (<8-mg sample for a 1.5 X 60-cm column in this case).

Homogeneity of F430, its isomeric derivatives, and F560 was determined by reversed-phase chromatography using either a PepRPC HR 5/5 column with the Pharmacia LKB Biotechnology Inc. Fast protein liquid chromatography system or a DeltaPak Cla column (3.9 X 15 mm) with the Waters 600 multisolvent delivery HPLC system (Waters, a Division of Millipore, Inc.). Flow rates of 0.7-1.0 ml/min were used for the analysis and a two-part linear gradient from 0 to 7% CH3CN in 9 ml and 7% to 16% CH3CN in 33 ml was found to be sufficient to separate the various isomers and derivatives of F130.

The dithionite reduction of FSW was carried out in a 50-ml ultrafiltration cell with a YC05 membrane (Amicon Division of W. R. Grace & Co., Danvers, MA). The solution containing homogeneous F, in 10 mM Tris/Cl- (pH 7) was bubbled with NZ in the capped ultrafiltration cell for -30 min. Upon the addition of 1.2 equivalents of Na2S204 under a steady stream of NP, with stirring, the dark purple solution immediately turned bright yellow. The solution was bubbled with N, in the capped ultrafiltration cell for an additional 20 min and then connected to a high pressure N, source to begin the ultrafiltration in order to remove excess dithionite and its reaction products. This yellow solution was subjected to 4 cycles of concentration to -3 ml followed by dilution with -50 ml of anoxic 18 megaohms-cm water. After the final concentration the yellow solution was lyophi- lized to dryness, dissolved in 10 mM Pi (pH 7.0) containing 25% (w/ w) glycerol, loaded into a Lucite/Mylar cell for XAS, and stored at 77 K until the spectrum was obtained.

The optical spectrum of the F430 obtained from the dithionite reduction of FSm indicated that it still contained -20% F5m, probably due to the reaction of trace amounts of oxygen with the excess dithionite, creatingperoxide (Creutz and Sutin, 1974), which we have found to be a facile oxidant of F430. The XAS data from this sample of F430 were also consistent with contamination by F560 and have therefore been corrected for this contamination by the subtraction of 20% of the spectrum of homogeneous F5m. Prior to obtaining the RR spectrum of the F430 prepared from the reduction of FSm, the sample was purified on a QAE-Sephadex A-25 column (1.5 X 60 cm) by elution with ammonium formate buffer (pH 4.2) according to published procedures (Shiemke et al., 1988a). As expected, the major contaminant in this sample was F5m. The F4s0 purified from this sample was shown to be homogeneous by reversed-phase HPLC and was chromatographically identical to F430 extracted directly from methyl reductase in high salt concentrations.

Samples for spectroscopy (XAS, RR, and optical) were prepared by taking an aliquot of purified F430 (or one of its derivatives) in water to dryness by lyophilization in a SpeedVac concentrator (Savant Instruments Inc., Farmingdale, NY). The freeze-dried sample was then dissolved in the desired buffer or solvent and stored frozen at 195 or 77 K until the spectra were obtained.

Spectroscopy-Optical absorption spectra were obtained with a Cary 219 spectrophotometer (Varian Instruments, Palo Alto, CA). Temperature was controlled by circulating water from a Lauda RC20 refrigerated circulator (Brinkman Instruments, Westbury, NY) through a jacketed cell holder. Resonance Raman spectra at ambient temperature were obtained on pairs of samples simultaneously, using a split cell designed for a Raman difference spectrometer described previously (Shelnutt, 1983). The spectrometer was not operated in the difference mode; spectra were collected on pairs of samples simultaneously. Low temperature (77 K) Raman spectra were ob-

11238 Coordination Chemistry of F430 TABLE I

X-ray absorption spectroscopic data collection and reduction for F430 and various derivatives Experimental parameter Edges EXAFS

SR facility SSRL SSRL Beamline VII-3 Monochromator crystal Si[400], Si[220]

11-2 (focused) Si[ l l l ]

Detection method Fluorescence Fluorescence Detector type Argon ion chambef Scan length, min 17

Argon ion chambef 24

Scans in average 3 10-20 Metal concentration, mM 8-12 8-12 Temperature, K 10,250,300 10,250,300 Energy standard Nickel foil (1st inflection) Nickel foil (1st inflection) Energy calibration, eV 8331.6 8331.6 Eo, eV 8350 8350 Pre-edge background

Spline background Energy range, eV (polynomial order) 8379-8730 (2)* 8380-8960 (2)*

Energy range, eV (polynomial order) 8375-8445 (2) 8374-8443 (2) 8445-8730 (2) 8443-8708 (3)

8708-8960 (3) EXAFS Co., Seattle, WA.

* Background was calculated from fitting this (EXAFS) region, then a constant subtracted so that the background matched the data just before the edge.

F m 0 FOOH 0

Native Fo. 13-monoepimer

FIG. 1. Non-crystallographically determined structures of F430, its configurational isomers, and the ring-oxidized derivative F,,o (adapted from Pfaltz et d., 1985).

tained on single samples frozen in 4-mm diameter NMR tubes contained in a liquid nitrogen filled EPR dewar with a transparent tail (Wilmad Inc., Buena, NJ). Spectra of the frozen samples were obtained in a backscattering geometry. Six to ten scans of each sample were collected and averaged to reduce the noise. Samples were 100- 200 @A in F430 and were excited with the 441.6 nm line of a helium- cadmium laser operating at -40 milliwatts, or the 406.7 or 413.1 nm lines of a krypton ion (Kr+) laser a t -100 milliwatts. Spectrometer resolution was 4 cm" in all cases, and all spectra were subjected to a fast-Fourier transform smoothing routine.

XAS data were collected at the Stanford Synchrotron Radiation Laboratory. Samples were maintained at 10 or 250 K during data collection using a custom designed (now Oxford Instruments CF1208) liquid helium cryostat. For data collection at ambient temperature the cryostat was not used and the sample temperature was not controlled. Reduction and analysis of the EXAFS data followed our standard protocol (Scott, 1985). Details of the data collection and reduction procedures are collected in Table I. For curve-fitting analysis, Ni-N scattering functions were extracted from the first shell of Ni(0Epc) (Ni-N = 1.909 A), and [Ni(corphin)SCN]z (Ni-N = 2.085

A) using complex Fourier back-transformation (Scott, 1985). Second- shell scattering functions were extracted in the same manner from Ni(0Epc) (Ni-C = 2.940 A). The Ni(0Epc) model complex was the kind gift of Dr. A. E. Eschenmoser (Eidgenossische Technische Hochschule, Zurich) and corresponds to complex 2 of Kratky et al. (1984). Published structural parameters (Kratky et al., 1985) were used for extraction of nickel EXAFS phase and amplitude functions from this model. The [Ni(corphin)SCN], model corresponds to the dimer of compound 1 of Kratky et at. (19841, and was also the kind gift of Dr. A. E. Eschenmoser. Structural parameters for this model were determined from atomic coordinates obtained from the Cam- bridge Crystallographic Data Base.

RESULTS AND DISCUSSION

Nature of the Axial Ligands in F43-When isolated from methyl reductase, F430 is susceptible to thermal isomerization and oxidative degradation (Diekert et al., 1981; Pfaltz et al., 1985). Through the use of NMR, nuclear Overhauser, and circular dichroism spectroscopies, the products of the isomerization have been identified as configurational isomers of F430, in which the relative stereochemical orientation of the side chains on pyrrolidine ring C (@-carbons C1, and C13, see Fig. 1) are reversed (Pfaltz et al., 1985). The initial product of this epimerization is the 13-monoepimer, which is irrevers- ibly converted to the 12,13-diepimer (Diekert et al., 1981). The conversion of F430 to the 12,13-diepimer is rather sluggish, having a half-life of -1 h at 80 "C and a half-life on the order of weeks at 4 "C. The product of the ring oxidation of F430 has been identified as 12J3-didehydr0-F~~~ (Pfaltz et al., 1985), in which a double bond exists between the B-carbons of pyrrolidine ring C (Fig. 1). This purple oxidation product is known as FSW, on the basis of its strong (t = 8300 M" cm-') absorption band at 560 nm (Pfaltz et al., 1985). An additional absorption band of similar intensity (t = 7500 M" cm") occurs at 475 nm. The oxidation reaction is also slow, occur- ring in aqueous solution in air at a rate comparable to that of the epimerization. The oxidation does appear to be autocata- lytic, proceeding faster in more concentrated F430 solutions. It has been reported that the nature of the solvent also affects the oxidation, with the rate of reaction following the order: acetic acid > methanol > water (Pfaltz et al., 1985). We have found that excess hydrogen peroxide will completely oxidize F430 to F,,, in a matter of minutes in aqueous solution at room temperature. The diepimer appears to be completely inert toward oxidation by air or peroxide.


In previous reports we presented evidence that the native isomer of F430 is 6-coordinate at 10 K in aqueous solution, whereas the configurational isomers are square-planar, 4- coordinate under the same conditions (Shiemke et al., 1988a, 1989). These conclusions are based primarily on comparison of their nickel K-edge x-ray absorption spectra. A pre-edge feature at 8336 eV is known to be characteristic of nickel in a square-planar geometry, whereas octahedral nickel complexes have a sharp, featureless edge rise (Eidsness et al., 1988). The 8336 eV shoulder is clearly present in the edge spectra of both the diepimer and the monoepimer obtained at 10 K, whereas native F430 at 10 K has the featureless edge characteristic of nickel in an octahedral geometry (Shiemke et al., 1988a, 1989). Similar conclusions regarding coordination number have been reached based on the room temperature magnetic moments of the isomers (Pfaltz et al., 1985). Analysis of the EXAFS data from these two complexes indicates that the different coordination geometries are accom- panied by other changes in the nickel coordination sphere. The native (non-epimerized) isomer has an expanded core with Ni-N bond distances of 2.1 A (Table 11, fit l ) , whereas the 4-coordinate monoepimer and diepimer have a covtracted macrocycle core, with Ni-N bond distances of -1.9 A (fits 3 and 5) (Shiemke et al., 1988a, 1989). It has been proposed that the macrocycle conformation is altered by the contraction of the core such that the largely planar macrocycle of F430 undergoes an S4 ruffling in the configurational isomers (Pfaltz et al., 1985; Eschenmoser, 1986), and results of our spectroscopic studies are in agreement with this proposal (Shiemke et al., 1988a). Similar ruffling is observed in the crystal structures of square-planar nickel complexes with highly reduced tetrapyrrole ligands (Kratky et al., 1985).

It has been reported that the oxidation of F430 can be reversed through the treatment of F660 with zinc dust in methanol solution (Pfaltz et al., 1985) or with dithionite in aqueous solution (Keltjens et al., 1983). The product of this ring reduction by zinc dust has been shown (by HPLC, optical, circular dichroism, 'H NMR, and mass spectroscopy) to be F430 in its native configuration, rather than the thermodynamically more stable diepimeric isomer. We have found that the dithionite reduction of F560 results in a product with resonance Raman and x-ray absorption spectra identical to

those of F430 that has been extracted from methyl reductase at high salt concentrations, implying that the axial ligands as well as the macrocycle conformations are the same.

The nickel x-ray absorption edge spectrum of F560 (Fig. 2a) has the pre-edge feature at 8336 eV and the qualitative shape characteristic of square-planar nickel complexes. Thus, F560 in dilute aqueous buffer contains 4-coordinate nickel in a square-planar coordination geometry. The EXAFS of F560 is consistent with this conclusion, being nearly identical to the EXAFS of the monoepimer and diepimer (not shown). Analy- sis of the F560 EXAFS (Table 11, fit 6) shows that the macrocycle of this deriyative has the contracted core (Ni-N bond distances of 1.88 A) found in the 4-coordinate configurational isomers of F430 (Shiemke et al., 1988a, 1989). After dithionite reduction of F560, the resultant F430 contains 6-coordinate, pseudo-octahedral nickel at 10 K in water. The x-ray absorption edge spectrum of the F430 derived from the reduction is identical to that of the freshly extracted cofactor (Fig. 2b). The spectra in the EXAFS region are also identical (Fig. 3), as are the room temperature RR spectra (Fig. 4). We have previously shown that the x-ray absorption and RR spectral characteristics are very sensitive to the nature of the axial ligands in F430 and its isomers (Shiemke et al., 1988a, 1988b, 1989). Thus, we can conclude that identical axial ligands are present in the F430 derived from the reduction of FHO and in the freshly extracted cofactor in aqueous solution. Further- more, since F560 contains 4-coordinate nickel prior to reduction, and is 6-coordinate after reduction of F430, the axial ligands must derive from the medium.

On the basis of these results, we propose that 6-coordinate F430 is a bis-aquo complex. Another possibility is that the axial ligands are provided by carboxylate groups from the macrocycle side chains through formation of dimers or higher aggregates. Both of these possibilities would be consistent with the finding that the axial ligands are provided by the medium and with the finding that the cofactor exists in an equilibrium distribution of 4- and 6-coordinate species near room temperature (see below). However, the room temperature optical spectrum of aqueous F430 is independent of cofactor concentration over a range of 5 p~ to 5 mM and unchanged over a pH range from 2 to 12 (not shown), indicating that aggregation of F430 does not occur to an appreciable extent in

TABLE I1 Curve-fitting results for the first and second shell of Fa, and its derivatives

Filter gives the portion of the FT that was isolated (using a half-Gaussian function of width 0.10 A) and back- transformed to k-space for curve fitting; N. is the coordination number per nickel; R is the nickel-scatterer distance; A 2 is a relative mean square deviation in R, AuZ = u2 (sample) - u2 (reference), where the reference compound is Ni(OEpc), unless otherwise noted. All samples were in 10 m M Pi (pH 7) with 20-25% (w/w) glycerol.

Sample Coordination Fit Ni-(O,N,C) Filter (A) shell NP R A 2 f ' Ref.

A A' ~~ ~ ~ ~~~

F130 First 1 1.29-2.12 6 2.10 0.0031 0.039 This work Second 2 1.29-3.17 8.0 3.03 0.0050 0.037 This work

Diepimer First 3 1.17-1.83 4 1.89 0.0001 0.032 Shiemke et al., 1989 Second 4 1.17-3.10 8.0 2.90 0.0050 0.042 Shiemke et al., 1989

Monoepimer First 5 1.15-1.88 4 1.89 -0.0020 0.060 Shiemke et al., 1989 FSSO First 6 1.10-1.88 4 1.88 -0.0005' 0.023 This work

Coordination numbers were not varied during optimization for the first shell fits. For the second-shell fits Au2

f ' is a relative goodness-of-fit statistic: was fixed while the number and distance of scatterers was varied.

where N is the number of data points to be fit. e The reference compound for this shell of scatterers is [Ni(corphin)SCNIa.

11240 Coordination Chemistry of F430

I 8300 8320 8340 8360 8380 8400

Energy (ev) FIG. 2. Nickel K-edge x-ray absorption spectra of: a, F560;

b, Fa30 obtained by extraction from methyl reductase (-), F430 obtained from the dithionite reduction of F560 (- - -). Spectra were obtained at 10 K, all samples are in 10 mM Pi (pH 7) with 25% (w/w) glycerol.

FIG. 3. Nickel K-edge EXAFS spectra of F430 obtained by extraction from methyl reductase (-); F430 obtained from the dithionite reduction of FBBO (- - -). Spectra were obtained at 10 K, and all samples are in 10 mM Pi (pH 7) with 25% (w/w) glycerol.

1631

r

a . 1314 w E

C

-

A

t I 900 1300 1700

Frequency (cm")

FIG. 4. Resonance Raman spectra at -305 K of F430 obtained by extraction from methyl reductase (a); F4~0 obtained by dithionite reduction of Fa60 (b ) . Both samples are in 25 mM P, (pH 7). The excitation wavelength was 413.1 nm.

aqueous solution. Thus, the axial ligands to F430 must be water molecules. Although we cannot specifically rule out hydroxide ligation, the greater prevalence of water molecules and the insensitivity of the F430 optical spectrum to pH argue against this possibility.

Analysis of the EXAFS of F430 also supports the assignment of the 6-coordinate form as the bis-aquo complex. We previously reported that filtered first-shell EXAFS of Frsa could be fit wiih six N or 0 donor ligands at an average distance of 2.10 A from the nickel (Table 11, fit l), and could not be fit with sulfur or higher-Z atoms (Shiemke et al., 1988a). This is consistent with either water or side-chain carboxylate axial ligation. These two types of axial ligands, in theory, can be distinguished by their different contribution to the second shell of scatters: water ligands would not contribute to the

second shell, whereas carboxylate ligands would each contribute one carbon to the second shell. The fitting procedure used for the second-shell analysis involves isolating the combined first- and second-shell FT peaks of F430, back-transforming to k-space, and fitting the resultant filtered EXAFS by holding the first-shell parameters fixed (fit 1) while varying the number and distance of second-shell carbons with a fixed Debye- Waller factor. The results (fit 2) show that the second shell of F430 consists of only the eight cu-carbons from the tetrapyrrole macrocycle at 3.03 A (this distance is also consistent with the macrocycle having a planar, expanded conformation). This result is consistent with bis-aquo ligation, but the difficulty in accurately determining coordination numbers from EXAFS analysis (especially from second-shell EXAFS) makes this a supportive, rather than a definitive, result.

Effect of Temperature on the Spectroscopic Properties of F43a-Resonance Raman spectral differences have been correlated with the different coordination geometries and macrocycle conformations exhibited by isomers of F430. In particular, the two highest energy peaks in the Raman spectra of the 4- coordinate monoepimer and diepimer (at -1530 and -1620 cm") are separated by 90 cm", whereas these peaks shift to -1545-1560 cm" and -1615-1630 cm" in 6-coordinate complexes of the cofactor (either epimeric or native isomer) resulting in a peak separation of -70 cm" (Shiemke et al., 1988b, 1989). Although the absolute frequencies of the peaks vary considerably among the different derivatives, the separation of the two high frequency peaks is found to be characteristic of the nickel coordination geometry (Shelnutt, 1987; Shiemke et al., 1988b, 1989). The RR spectrum of F430 at room temperature (-295 K) contains features characteristic of both the 4-coordinate ruffled and the 6-coordinate planar macrocycle conformations (Fig. 5). Our previous proposal that this phenomenon is due to the dynamic axial ligation equilibrium between 4- and 6-coordinate forms of F430 in aqueous solution (Shiemke et al., 1988b) is now confirmed by demonstration of the temperature dependence of the Raman, optical, and x-ray absorption spectra of F430.

The RR spectrum of F430 at 77 K differs markedly from the room temperature spectrum. The 1534 cm" feature that we have assigned to the 4-coordinate form of the cofactor (Shiemke et al., 1988b) is absent at low temperature (Fig. 5),

1280 1360 1440 1520 1600 1680 Frequency (Cm")

FIG. 5. Resonance Raman spectra of F430 in 25 mM PI (pH 7) at -295 K obtained with 406.7 nm excitation (a), 413.1 nm excitation (b ) , 441.6 nm excitation (c) , and at 77 K with 441.6 nm excitation (d) .


leaving only the 1555 cm" peak of the 6-coordinate form, now assigned to the bis-aquo complex. The separation of the two highest energy Raman features (at 1555 and 1629 cm") is 74 cm" in the 77 K spectrum, similar to the -70 cm" separation of the analogous peaks in the room temperature RR spectra of 6-coordinate cyanide, pyridine, or l-methylimidazole complexes of F430 and its isomers (Shiemke et al., 1989). The high frequency Raman spectra of a nickel-corphin complex with a chromophore structure nearly identical to that of F430 are very similar to the F430 Raman spectra and exhibit an -70 cm" separation of these peaks in 6-coordinate complexes (Shelnutt, 1987; Shiemke et al., 1988b). These temperature-dependent changes in the F430 RR spectrum are completely reversible: warming the frozen sample to room temperature after recording the low temperature spectrum yields the original room temperature spectrum. Thus, irreversible processes such as epimerization or oxidation are not responsible for this temperature-dependent spectral change, and we attribute this temperature dependence to the equilibrium illustrated in Equation 1.'

F430 + 2H20 + F4m(H20)2, K' e /%[Hz01 - - [F~o(HzO)zl (1)

[Fa01

The effect of temperature on the x-ray absorption spectrum of aqueous F430 also supports the existence of the equilibrium shown in Equation 1. The x-ray absorption spectrum of aqueous F430 at 10 K (Fig. 6a) has the sharp, featureless edge characteristic of nickel in a pseudo-octahedral coordination geometry (Eidsness et al., 1988, Shiemke et al., 1988a). At -298 K, the edge spectrum of FdS0 displays a marked shoulder at -8336 eV and a somewhat broadened and flattened peak (Fig. 6a). This high temperature spectrum is precisely what one would expect from a mixture of square-planar and pseudo- octahedral nickel complexes. As with the Raman data, these changes in the x-ray absorption edge spectrum are completely reversible: the edge spectra acquired at 10 K before and after the room temperature experiment are identical. As a control, we have also recorded the x-ray absorption edge spectrum of the diepimer at room temperature and at 10 K. As shown in Fig. 6b, the spectrum of this isomer is characteristic of square- planar nickel and independent of temperature from 10 to -300 K (aside from some thermal broadening), as expected.

The nickel EXAFS spectrum of F430 is also affected by temperature. We could not obtain EXAFS data for F430 at room temperature, due to the likelihood of epimerization or oxidation of the cofactor taking place over the -4 h needed for collection of EXAFS data. However, these irreversible reactions are slowed down enough at 250 K that collection of EXAFS data without their interference is possible. Relative to the 10 K data, the amplitude of the EXAFS oscillation at 250 K is markedly reduced in the region from k = 3-10 A-' (Fig. 7a). This reduced EXAFS amplitude is reflected in the decreased intensity of the first-shell FT peak at 250 K, relative to 10 K (Fig. 7b). The reduced EXAFS amplitude at 250 K can be explained by a mixture of 4- and 6-coordinate species in solution at this temperature, due to the equilibrium of Equation 1. The EXAFS oscillations from k = 3-10 A" of the square-planar diepimer are -180" out of phase with the EXAFS of the pseudo-octahedral complexes, due to the- dif- ference in average Ni-N bond distances (-1.9 and -2.1 A for the 4- and 6-coordinate species, respectively). Thus, the presence of a small amount of 4-coordinate species in aqueous

'The equilibrium constant for this overall reaction (0') is the product of the equilibrium constants for the stepwise addition of the two axial water molecules. K' is defined such that the concentration of water is not explicitly taken into account.

I 830083208340836083808400

Energy (eV) FIG. 6. a, nickel K-edge x-ray absorption spectra of F4m at 10 K

of the 12J3-diepimer of F430 at 10 K (-), and at -300 K (- - -). All (-), and at -300 K (- - -), b, nickel K-edge x-ray absorption spectra

samples were in 10 mM Pi (pH 7), with 25% (w/w) glycerol.

-$!O 4.8 6:s 8.4 10.2 12.0 k (A")

18 b

FIG. 7. EXAFS (a) and Fourier transforms (b ) of Faso at 10 K (-), and at 260 K (- - -). Both samples were in 10 mM Pi (pH 7), with 25% (w/w) glycerol.

solution at 250 K would serve to damp out the EXAFS of the predominant bis-aquo complex. This requires that the 4- coordinate form of native F430 have short (-1.9 A) Ni-N bonds to get the phase relationship necessary for the damping of the EXAFS amplitude. It would appear, therefore, that the 4- coordinate form of native F430 has a contracted (and probably ruffled) macrocycle conformation similar to that of its diepimeric and monoepimeric isomers, as has been suggested by Eschenmoser and coworkers (Pfaltz et al., 1985). The features attributed to the 4-coordinate species of F430 in the room temperature Raman spectrum (Fig. 5) are similar to those of the configurational isomers (Shiemke et al., 198813, 1989), further supporting the structural similarity of the 4-coordinate conformation in all three isomers of F430

We have previously demonstrated that characteristic changes in the optical spectrum accompany the axial binding of cyanide, pyridine and 1-methylimidazole (Shiemke et al., 1989). Since aqueous solutions of native F430 contain both 4- and 6-coordinate species in equilibrium, the optical spectrum should vary with temperature near 300 K. This is indeed the case, as demonstrated in Fig. 8. The changes in the optical spectrum illustrated in Fig. 8 are completely reversible. The presence of isosbestic points at -285, -365, and -445 nm also indicates that only two species are present that are readily interconverted. Thus, the 5-coordinate intermediate species does not occur to any appreciable extent. At 275 K we expect relatively more of the bis-aquo complex than at 300 K, and

Coordination Chemistry of F430 1 7

T ( O K )

275

293

31 3

333

300 400 500

Wavelength (nm) FIG. 8. Temperature dependence of the optical spectrum of

F430. The sample was in 50 mM ammonium formate buffer (pH 7.6). Isosbestic points are indicated by arrows.

2.0

1 .O

c Y C v -

0.0

-1 .o

3.0 3.5 4.0

I/T X 103 FIG. 9. Van't Hoff plot for the aquation of F4~0. The closed

circles are from the optical spectra and the open circles are from XAS measurement^.^

features are characteristic of the contracted macrocycle conformation of the 4-coordinate monoepimer and diepimer and disappear upon formation of 6-coordinate pseudo-octahedral complexes (Shiemke et al., 1988a, 1989).

Additional evidence of these assignments of the optical spectrum comes from the dependence of the Raman spectrum on excitation wavelength. The RR spectra shown in Fig. 4 were acquired with 413.1 nm excitation, close to the peak maximum (421 nm) attributed to the 6-coordinate form of F430. Thus, we would expect that this excitation wavelength should preferentially enhance the 1556-cm" Raman peak of the 6-coordinate form of F430. When the excitation wavelength is moved further to the red (441.6 nm, Fig. 5c) the intensity of the 1534-cm" peak due to the square-planar complex increases at the expense of the 1556-cm" peak, whereas excitation to the blue of 413.1 nm (406.7 nm, Fig. 5a) results in enhancement of the 1556-cm" peak of the bis-aquo complex. This indicates that the bis-aquo complex of F430 is responsible for the 421-nm feature in the optical spectrum, whereas the 4-coordinate species is associated with the 434- nm peak.

It has been reported that F430 in water has a room temperature magnetic moment ( p ) of 2.0 * 0.2 Bohr magnetons (BM) (Pfaltz et al., 1985). This moment is much lower than expected for octahedral (or 5-coordinate) nickel(I1) complexes (which typically have p = 2.9-3.4 BM), but much higher than the value of zero expected for diamagnetic square-planar nickel complexes (Cotton and Wilkinson, 1980). Such an intermediate magnetic moment is, however, consistent with an equilibrium mixture of diamagnetic square-planar and paramagnetic ( p = 3 BM) octahedral forms of F430 at room temperature in aqueous solution. A similar room temperature moment ( p = 2.1 BM) is observed for aqueous Ni(tetra-N-4- methylpyridyl porphine) which exists as a mixture of the square-planar and bis-aquo octahedral species (Pasternack et al., 1974).

The reaction proceeding to the right in Equation 1 involves the formation of bonds between the nickel of F430 and the axial water ligands and would therefore be expected to have a negative standard enthalpy of reaction (AH"). The van't Hoff equation (Equation 2)2

ln(K') = - -AH" ASo RT R

+-

predicts that if AH' is negative, the equilibrium constant will increase as the temperature decreases. Reducing the temperature will, therefore, drive the ligand-binding reaction to the right and increase the relative amount of the 6-coordinate bis-aquo complex. This is precisely what is observed in all the temperature-dependent spectroscopic measurements of F430. Combining quantitative estimates of the proportions of 4- and 6-coordinate forms of F430 at various temperatures from optical and XAS measurements3 allows the estimation of AH"

we observe that a peak at -421 nm is predominant at this temperature (Fig. 8). At 333 K the equilibrium favors the 4- coordinate species, and a peak at -434 nm is observed. The spectrum of aqueous F430 that is normally presented in the literature (Hausinger et al., 1984; Livingston et al., 1984; Shiemke et al., 1988a) is obtained at -295 K and is therefore a mixture of the 421 nm feature of the bis-aquo complex and the 434 nm peak of the 4-coordinate moiety (Fig. 8). Other changes in the optical spectrum are consistent with these assignments. As temperature is increased from 275 to 333 K, shoulders at -295, 350, and perhaps -500 nm appear. These

The relative proportions of the 4- and 6-coordinate species at room temperature were determined by mimicking the room temperature edge spectrum (Fig. 6a) with a weighted average of the 10 K edge spectrum of FGO and the room temperature edge spectrum of the diepimer. From this value of K' (l.O), the molar absorptivities of the two species were determined and these molar absorptivities were then used to determine K' from the spectra displayed in Fig. 8. The fits of the room temperature edge spectrum were not exact probably due to thermal broadening of the spectrum of the 6-coordinate species relative to the 10 K FGo spectrum. Acceptable fits yielded a range of K' from 0.85 to 1.10 with the best fit yielding K' = 1.0. The uncertainties in AH' and AS" stem predominantly from the errors introduced by this fitting procedure.


= -23 k 8 kJ . mol" and A S o = -74 k 25 J. mol". K" (Fig. 9).

Thermodynamic parameters of similar magnitude have been reported for the solid-phase square-planar to octahedral conversion of several nickel-bis(l,2-cyclohexanediamine) complexes (Ihara et al., 1987). The small magnitude of A H o

for these latter reactions was attributed to a close balance between the exothermic contribution of the axial-ligand bond formation and the endothermic contribution from the weak- ening of the equatorial metal-ligand bonds and the subsequent reorganization of these ligands. For the aquation of the Ni(tetra-N-4-methylpyridyl porphine), more negative values of A H o and ASo are reported (-39 kJ.mol", and -130 J . mol".K", respectively) (Pasternack et al., 1974). This may indicate that the rigid porphyrin does not require as much energetically unfavorable macrocycle reorganization as occurs in F430.

Axial Ligation Equilibria in Other Solvents-The equilibrium distribution of 4- and 6-coordinate forms of F430 in aqueous solution implies that the nickel has a low affinity for axial water ligands. This affinity is much smaller than for cyanide, pyridine, and imidazoles which form fully 6-coordinate complexes at room temperature (Shiemke et al., 1989). F430 also shows low affinity for alcoholic ligands similar to water, such that both 4- and 6-coordinate species are observed in methanol and ethanol. The presence of both the 4-coordinate and bis-solvent forms can be observed in the optical spectra of F430 in these alcoholic solvents. The peak maxima for the -430 nm feature occur at 431 nm in methanol and at 435 nm in ethanol (Fig. 10, b and c). In both these solvents a shoulder is observed at -420 nm, similar to the feature we have attributed to the bis-aquo form of F430 in water at room temperature (Fig. loa). The intensity of the -420 nm shoulder is much smaller in ethanol than in methanol, suggesting that ethanol is a weaker ligand than methanol. On the basis of the intensity of this shoulder it also appears that the bis-solvent complex of F430 is more prevalent in water than in methanol at 298 K. The concentration of water molecules is 55 M in aqueous solution, much higher than the concentration of methanol molecules in neat methanol (24.5 M), and this probably accounts for the relatively greater prevalence of the

I I\\

300 400 500

Wavelength (nm) FIG. 10. Optical absorption spectra of F43o at 298 K in: a,

50 mM ammonium formate (pH 7.6); b, neat methanol; c, neat ethanol; d, neat 2,2,2-trifluoroethanol.

bis-aquo complex. The magnetic moment of F430 in methanol has been reported as 1.8 BM (Pfaltz et al., 19851, slightly lower than in water, consistent with a smaller ratio of 6- coordinate to 4-coordinate forms in methanol.

The temperature dependence of the optical spectra of F430

in methanol and ethanol are consistent with the assignment of the -420 nm shoulder to a bis-solvent complex. In both of these solvents the intensity of this shoulder (and the full width at half-maximum of the visible absorption band) increases as the temperature decreases (not shown). The room temperature RR spectra of F430 in methanol and ethanol exhibit peaks at -1534 and -1556 cm" (not shown), similar to the spectrum in aqueous solution and consistent with a mixture of 4- and 6-coordinate species. The ratio of 6-coordinate to 4-coordinate species determined from the relative intensities of the Raman peaks in the different solvents is analogous to the interpretation of the optical spectra, with the relative concentration of the bis-solvent complex following the order: water > methanol > ethanol.

The trifluoromethyl substituent on 2,2,2-trifluoroethanol renders the alcohol oxygen of this solvent a very poor coordinating group. We would, therefore, expect F430 to be predominantly (if not completely) 4-coordinate in this solvent. The optical spectrum of F430 in 2,2,2-trifluoroethanol (Fig. 10d) is fully consistent with this, as is the fact that the 13C NMR spectrum of F430 could be observed in this solvent (Livingston et al., 1984). The optical spectrum has shoulders at -500, -360, and -295 nm, characteristic of the contracted, ruffled macrocycle conformation in the 4-coordinate epimeric isomers of F430 (Shiemke et al., 1988a, 1989). Furthermore, the peak maximum occurs at 438 nm in 2,2,2-trifluoroethanol and lacks the -420 nm shoulder, similar to the spectra of F430 in aqueous solution at 333 K and in ethanol at 298 K (Figs. 8 and lOc), under which conditions the cofactor is predominantly 4-coordinate. A similar optical spectrum is observed for F430 in 2-mercaptoethanol (not shown), which also has a strong electron-withdrawing group (the thiol moiety) and should therefore have a weakly coordinating alcohol oxygen. The pentamethyl ester of F430 (F43OM) is reported to be diamagnetic (and therefore 4-coordinate) in methylene chloride (Pfaltz et al., 1985) and exhibits a spectrum similar to that of F430 in 2,2,2-trifluoroethanol. As expected, the RR spectra of F430 in 2,2,2-trifluoroethanol and 2-mercaptoethanol have features of only the 4-coordinate form of the cofactor (not shown). The spectral changes observed for F430 in 2,2,2-trifluoroethanol and 2-mercaptoethanol are completely reversible (removal of these solvents followed by dissolution in water yields the original spectrum) and are, therefore, not due to accelerated epimerization in these solvents.

The above interpretation of the spectra of F430 in water, methanol, and ethanol indicates that the native isomer of F430

has a small, but observable, affinity for such weak field oxygen donor ligands. In contrast, the configurational isomers of F430 (the 13-monoepimer and the 12,13-diepimer) show essentially no affinity for these ligands. The x-ray absorption edge spectrum of the diepimer in water is characteristic of 4-coordinate nickel at 10 and 300 K (Fig. 6b), as are its RR and optical spectra at 300 K (Shiemke et al., 1988a, 1988b, 1989). The room temperature RR and optical spectra of the diepimer in methanol and ethanol are nearly identical to the spectra in water, indicating that these solvents do not coordinate at room temperature (XAS data indicate that methanol will coordinate below 150 K, not shown). Similar spectra for the monoepimer indicate that it too has no affinity for water or alcohol moieties as axial ligands at room temperature. All these isomers of F430 will bind cyanide, pyridine, and imidaz-

11244 Coordination Chemistry of F430

oles, and we have shown that the macrocycle conformations of these 6-coordinate complexes are very similar and are apparently independent of the configuration of the ring C side chains (Shiemke et al., 1989). It appears (on the basis of limited XAS and RR data) that the macrocycle conformation of the 4-coordinate form of the native isomer of F430 is very similar to the conformation displayed by the 4-coordinate configurational isomers. Thus, the differences among these isomers in affinity for axial ligands must stem from thermodynamic consequences of subtle structural differences.

Eschenmoser and coworkers (Pfaltz et al., 1985; Kratky et al., 1985; Eschenmoser, 1986) have proposed that steric inter- action between the side chain of C13 and the carbonyl oxygen of the nearby carbocyclic ring (cf. Fig. 1) makes the contracted, ruffled macrocycle conformation unfavorable for native F430 and that this steric constraint is removed by epimerization. Our spectroscopic studies provide direct evidence that the native isomer is capable of adopting a contracted (and presumably ruffled) conformation similar to the epimerized species, but this conformation is indeed less favorable energetically for F430 than for its configurational isomers. We can rationalize this finding by proposing that the steric constraint in the native cofactor results in a smaller difference in energy between the contracted (4-coordinate) and expanded (6-coordinate) conformations of native F430 compared to the analogous energy difference in the configurational isomers. For the native isomer, the relatively small thermodynamic advan- tage gained through formation of bonds to weak-field axial ligands (e.g. water, methanol, ethanol) would be comparable to the energy needed to generate the expanded, planar conformation of the macrocycle. The contracted, ruffled conformation of the macrocycle in the diepimer is thermodynamically more stable than either the ruffled or planar conformations of the native cofactor, leading to the irreversibility of the conversion from the native isomer to the diepimer, and the relatively greater difficulty of forming 6-coordinate complexes of the diepimer. Octahedral complexes of the diepimer are formed only when the thermodynamically unfavorable core expansion and macrocycle planarization can be compen- sated for by the formation of bonds to strong-field axial ligands with the capability of n-back bonding (e.g. cyanide, pyridine, and imidazoles) (Shiemke et al., 1989).

Implications for the Structure and Function of Enzyme- bound F430-The pseudo-octahedral complexes of F430 with water, methanol, and ethanol contribute a feature at -420 nm to the room temperature optical spectra (Fig. 10, a-c). This is in sharp contrast to the optical spectra of the complexes of FAZ0 with cyanide, pyridine, and 1-methylimidazole, which have peak maxima at 433-439 nm (Shiemke et al., 1989). The lower energy of the transition in the complexes with the strong-field nitrogen-donor ligands may be due to n- back bonding between the metal and axial ligands lowering the energy of the a* excited electronic state of the macrocycle. However, the optical spectra of the complexes with the non- n-bonding water, methanol, or ethanol ligands more closely resemble the spectrum of the enzyme-bound cofactor, which has a peak maximum at 420 nm and a shoulder at -445 nm (Hausinger et al., 1984). This suggests that the endogenous axial ligands are more like water or alcohol oxygen than the unsaturated nitrogen of imidazole. However, the RR spectra of F430 with these weak-field ligands to not approximate the spectrum of enzyme-bound F430 (Shiemke et al., 198813,19891, although the RR spectral features are due to vibrations of the macrocycle, making this technique more sensitive to altera- tions in the conformation of the chromophore and only indi- rectly characteristic of the nature of the axial ligand. Thus,

the unique RR spectrum of enzyme-bound F430 may reflect a unique macrocycle conformation caused by specific interac- tions with the protein binding site.

Our results support the conclusion that the conformation of the macrocycle has profound effects on the coordination chemistry of the nickel in F430. The stereochemical configuration of the pyrrolidine ring C substituents in the native cofactor (Fig. 1) induces a macrocycle conformational pref- erence that enhances the affinity of the nickel for axial ligands. Since it is only the native isomer of F430 that is incorporated into methyl reductase to generate active enzyme (Hartzell and Wolfe, 1986), it can be postulated that this macrocycle-based stereochemical control of nickel-ligand binding affinity plays an important physiological role. The strong temperature dependence of this binding affinity may also be physiologically relevant, since the methyl reductase enzyme is isolated from a thermophilic bacterium with an optimum growth temperature of 65 "C. In vitro studies of enzyme activity indicate that the components of the methane production assay can be combined at room temperature with activity being observed only after heating to -60 "C (Ellefson et al., 1982). Such a temperature may be required to facilitate dissociation of endogenous nickel axial ligands so that substrate (CH3SCoM or HSHTP) binding can occur. We are currently studying the spectroscopic properties of enzyme- bound F430 to determine if structural changes accompany the thermal activation of the methyl reductase enzyme.

Acknowledgments-We would like to thank Dr. Ralph S. Wolfe for the generous gift of the Methunobacterium cells. We also thank Dr. Marly K. Eidsness for contributing to the early stages of this inves- tigation and Dr. A. Eschenmoser for his many helpful comments during the review process. The XAS data were collected at the Stanford Synchrotron Radiation Laboratory (SSRL), which is funded by the Department of Energy under Contract DE-AC03-82ER-13000, Office of Basic Energy Sciences, Division of Chemical Sciences and the National Institutes of Health, Biotechnology Resource Program, Division of Research Resources. XAS studies (under R. A. S.) at Georgia are supported by National Science Foundation Grant DMB 86-45819 (formerly DMB 85-02707).

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coordination chemistry of f430 - journal of biological chemistry

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