Biomedical subjects

C D Stout

Publications and source records attributed to C D Stout.

At least 37 records · Page 2Linked to original sources

PubMed1998

A T14C variant of Azotobacter vinelandii ferredoxin I undergoes facile [3Fe-4S]0 to [4Fe-4S]2+ conversion in vitro but not in vivo.

[4Fe-4S]2+/+ clusters that are ligated by Cys-X-X-Cys-X-X-Cys sequence motifs share the general feature of being hard to convert to [3Fe-4S]+/0 clusters, whereas those that contain a Cys-X-X-Asp-X-X-Cys motif undergo facile and reversible cluster interconversion. Little is known about the factors that control the in vivo assembly and conversion of these clusters. In this study we have designed and constructed a 3Fe to 4Fe cluster conversion variant of Azotobacter vinelandii ferredoxin I (FdI) in which the sequence that ligates the [3Fe-4S] cluster in native FdI was altered by converting a nearby residue, Thr-14, to Cys. Spectroscopic and electrochemical characterization shows that when purified in the presence of dithionite, T14C FdI is an O2-sensitive 8Fe protein. Both the new and the indigenous clusters have reduction potentials that are significantly shifted compared with those in native FdI, strongly suggesting a significantly altered environment around the clusters. Interestingly, whole cell EPR have revealed that T14C FdI exists as a 7Fe protein in vivo. This 7Fe form of T14C FdI is extremely similar to native FdI in its spectroscopic, electrochemical, and structural features. However, unlike native FdI which does not undergo facile cluster conversion, the 7Fe form T14C FdI quickly converts to the 8Fe form with a high efficiency under reducing conditions.

Amino Acid Sequence↗

PubMed1998

Structure of Azotobacter vinelandii 7Fe ferredoxin at 1.35 A resolution and determination of the [Fe-S] bonds with 0.01 A accuracy.

The crystal structure of Azotobacter vinelandii ferredoxin I (FdI) at 100 K has been refined at 1.35 A resolution by full matrix block diagonal least-squares methods with anisotropic temperature factors for all non-hydrogen atoms and with hydrogen atoms included in the model. Fe-S bonds within the [3Fe-4S]+ and [4Fe-4S]2+ clusters of the protein are determined with an accuracy of at least 0.01 A. Analysis of metric parameters reveals greater variation in bonds and angles within the [3Fe-4S]+ cluster than in the [4Fe-4S]2+ cluster, whereas the opposite is true regarding the cysteine Sgamma atoms ligating to the two [Fe-S] cores. The [3Fe-4S]+ core is asymmetrically distorted by the protein matrix but relatively uniformly ligated by its three Cys ligands; in contrast the tetrahedral [4Fe-4S]2+ core is relatively symmetric but non-uniformily ligated by its four Cys ligands, three of which occur in a conserved CysxxCysxxCys residue motif. Comparison of the [3Fe-4S]+ clusters in FdI and Desulfovibrio gigas ferredoxin II, refined at 1.7 A resolution, indicates that within the limit of accuracy of the two refinements the cuboidal core is differently distorted in the two proteins. Comparison of the [3Fe-4S]+ core in FdI with the structure of a reduced [Fe3S4]o synthetic analog indicates that the protein-bound cluster displays distortions not intrinsic to the core itself. Nevertheless, both [3Fe-4S]+ and [Fe3S4]o cores have metric features consistent with expected trends due to net charge on Fe and valency of S, and both exhibit a splayed configuration with respect to their three mu2S atoms in the absence of a fourth Fe. Comparison of the [4Fe-4S]2+ cluster in FdI with the structures of [Fe4S4]2+ synthetic analogs shows that the protein bound and synthetic cubanes are very similar in geometric parameters, including the presence of tetragonal distortion in the FdI cluster common to this oxidation state.

Azotobacter vinelandii↗

PubMed1998

The crystal structure of NADPH:ferredoxin reductase from Azotobacter vinelandii.

NADPH:ferredoxin reductase (AvFPR) is involved in the response to oxidative stress in Azotobacter vinelandii. The crystal structure of AvFPR has been determined at 2.0 A resolution. The polypeptide fold is homologous with six other oxidoreductases whose structures have been solved including Escherichia coli flavodoxin reductase (EcFldR) and spinach, and Anabaena ferredoxin:NADP+ reductases (FNR). AvFPR is overall most homologous to EcFldR. The structure is comprised of a N-terminal six-stranded antiparallel beta-barrel domain, which binds FAD, and a C-terminal five-stranded parallel beta-sheet domain, which binds NADPH/NADP+ and has a classical nucleotide binding fold. The two domains associate to form a deep cleft where the NADPH and FAD binding sites are juxtaposed. The structure displays sequence conserved motifs in the region surrounding the two dinucleotide binding sites, which are characteristic of the homologous enzymes. The folded over conformation of FAD in AvFPR is similar to that in EcFldR due to stacking of Phe255 on the adenine ring of FAD, but it differs from that in the FNR enzymes, which lack a homologous aromatic residue. The structure of AvFPR displays three unique features in the environment of the bound FAD. Two features may affect the rate of reduction of FAD: the absence of an aromatic residue stacked on the isoalloxazine ring in the NADPH binding site; and the interaction of a carbonyl group with N10 of the flavin. Both of these features are due to the substitution of a conserved C-terminal tyrosine residue with alanine (Ala254) in AvFPR. An additional unique feature may affect the interaction of AvFPR with its redox partner ferredoxin I (FdI). This is the extension of the C-terminus by three residues relative to EcFldR and by four residues relative to FNR. The C-terminal residue, Lys258, interacts with the AMP phosphate of FAD. Consequently, both phosphate groups are paired with a basic group due to the simultaneous interaction of the FMN phosphate with Arg51 in a conserved FAD binding motif. The fourth feature, common to homologous oxidoreductases, is a concentration of 10 basic residues on the face of the protein surrounding the active site, in addition to Arg51 and Lys258.

Azotobacter vinelandii↗

PubMed1997

Y13C Azotobacter vinelandii ferredoxin I. A designed [Fe-S] ligand motif contains a cysteine persulfide.

Ferredoxins that contain [4Fe-4S]2+/+ clusters often obtain three of their four cysteine ligands from a highly conserved CysXXCysXXCys sequence motif. Little is known about the in vivo assembly of these clusters and the role that this sequence motif plays in that process. In this study, we have used structure as a guide in attempts to direct the formation of a [4Fe-4S]2+/+ in the [3Fe-4S]+/0 location of native (7Fe) Azotobacter vinelandii ferredoxin I (AvFdI) by providing the correct three-dimensional orientation of cysteine ligands without introducing a CysXXCysXXCys motif. Tyr13 of AvFdI occupies the position of the fourth ligating cysteine in the homologous and structurally characterized 8Fe ferredoxin from Peptococcus aerogenes and a Y13C variant of AvFdI could be easily modeled as an 8Fe protein. However, characterization of purified Y13C FdI by UV-visible spectra, circular dichroism, electron paramagnetic resonance spectroscopies, and by x-ray crystallography revealed that the protein failed to use the introduced cysteine as a ligand and retained its [3Fe-4S]+/0 cluster. Further, electrochemical characterization showed that the redox potential and pH behavior of the cluster were unaffected by the substitution of Tyr by Cys. Although Y13C FdI is functional in vivo it does differ significantly from native FdI in that it is extremely unstable in the reduced state possibly due to increased solvent exposure of the [3Fe-4S]0 cluster. Surprisingly, the x-ray structure showed that the introduced cysteine was modified to become a persulfide. This modification may have occurred in vivo via the action of NifS, which is known to be expressed under the growth conditions used. It is interesting to note that neither of the two free cysteines present in FdI was modified. Thus, if NifS is involved in modifying the introduced cysteine there must be specificity to the reaction.

Amino Acid Sequence↗

PubMed1996

The reaction of fluorocitrate with aconitase and the crystal structure of the enzyme-inhibitor complex.

It has been known for many years that fluoroacetate and fluorocitrate when metabolized are highly toxic, and that at least one effect of fluorocitrate is to inactivate aconitase. In this paper we present evidence supporting the hypothesis that the (-)-erythro diastereomer of 2-fluorocitrate acts as a mechanism based inhibitor of aconitase by first being converted to fluoro-cis-aconitate, followed by addition of hydroxide and with loss of fluoride to form 4-hydroxy-trans-aconitate (HTn), which binds very tightly, but not covalently, to the enzyme. Formation of HTn by these reactions is in accord with the working model for the enzyme mechanism. That HTn is the product of fluorocitrate inhibition is supported by the crystal structure of the enzyme-inhibitor complex at 2.05-A resolution, release of fluoride stoichiometric with total enzyme when (-)-erythro-2-fluorocitrate is added, HPLC analysis of the product, slow displacement of HTn by 10(6)-fold excess of isocitrate, and previously published Mössbauer experiments. When (+)-erythro-2-fluorocitrate is added to aconitase, the release of fluoride is stoichiometric with total substrate added, and HPLC analysis of the products indicates the formation of oxalosuccinate, and its derivative alpha-ketoglutarate. This is consistent with the proposed mechanism, as is the formation of HTn from (-)-erythro-2-fluorocitrate. The structure of the inhibited complex reveals that HTn binds like the inhibitor trans-aconitate while providing all the interactions of the natural substrate, isocitrate. The structure exhibits four hydrogen bonds < 2.7 A in length involving HTn, H2O bound to the [4Fe-4S] cluster, Asp-165 and His-167, as well as low temperature factors for these moieties, consistent with the observed very tight binding of the inhibitor.

Aconitate Hydratase↗

PubMed1996

Crystallization of ADP-ribosyl cyclase from Aplysia californica.

ADP-ribosyl cyclase synthesizes the secondary messenger cyclic ADP-ribose from NAD+. Diffraction quality crystals of the enzyme from ovotestes of Aplysia californica have been obtained. Crystallographic analysis of this enzyme will yield insight into the mode of binding of the novel cyclic nucleotide and the mechanism by which NAD+ is cyclized.

ADP-ribosyl Cyclase↗

PubMed1996

Crystal structure of dUTP pyrophosphatase from feline immunodeficiency virus.

We have determined the crystal structure of dUTP pyrophosphatase (dUTPase) from feline immunodeficiency virus (FIV) at 1.9 A resolution. The structure has been solved by the multiple isomorphous replacement (MIR) method using a P6(3) crystal form. The results show that the enzyme is a trimer of 14.3 kDa subunits with marked structural similarity to E. coli dUTPase. In both enzymes the C-terminal strand of an anti-parallel beta-barrel participates in the beta-sheet of an adjacent subunit to form an interdigitated, biologically functional trimer. In the P6(3) crystal form one trimer packs on the 6(3) screw-axis and another on the threefold axis so that there are two independent monomers per asymmetric unit. A Mg2+ ion is coordinated by three asparate residues on the threefold axis of each trimer. Alignment of 17 viral, prokaryotic, and eukaryotic dUTPase sequences reveals five conserved motifs. Four of these map onto the interface between pairs of subunits, defining a putative active site region; the fifth resides in the C-terminal 16 residues, which is disordered in the crystals. Conserved motifs from all three subunits are required to create a given active site. With respect to viral protein expression, it is particularly interesting that the gene for dUTPase (DU) resides in the middle of the Pol gene, the enzyme cassette of the retroviral genome. Other enzymes encoded in the Pol polyprotein, including protease (PR), reverse transcriptase (RT), and most likely integrase (IN), are dimeric enzymes, which implies that the stoichiometry of expression of active trimeric dUTPase is distinct from the other Pol-encoded enzymes. Additionally, due to structural constraints, it is unlikely that dUTPase can attain an active form prior to cleavage from the polyprotein.

Amino Acid Sequence↗

PubMed1996

Crystal structure of Aplysia ADP ribosyl cyclase, a homologue of the bifunctional ectozyme CD38.

ADP ribosyl cyclase synthesizes the novel secondary messenger cyclic ADP ribose (cADPR) utilizing NAD as a substrate. The enzyme shares extensive sequence similarity with two lymphocyte antigens, CD38 and BST-1, which hydrolyse as well as synthesize cADPR. The crystal structure provides a model for these cell surface enzymes. Cyclase contains two spatially separated pockets composed of sequence conserved residues, suggesting that the cyclization reaction may entail use of distinct sites. The enzyme dimer encloses a cavity which may entrap the intermediate, ADP ribose.

ADP-ribosyl Cyclase↗

PubMed1995

Site-directed mutagenesis of Azotobacter vinelandii ferredoxin I: cysteine ligation of the [4Fe-4S] cluster with protein rearrangement is preferred over serine ligation.

The [4Fe-4S] cluster of Azotobacter vinelandii ferredoxin I receives three of its four ligands from a Cys-Xaa-Xaa-Cys-Xaa-Xaa-Cys sequence at positions 39-45 while the fourth ligand, Cys20, is provided by a distal portion of the sequence. Previously we reported that the site-directed mutation of Cys20 to Ala (C20A protein) resulted in the formation of a new [4Fe-4S] cluster that obtained its fourth ligand from Cys24, a free cysteine in the native structure. That ligand exchange required significant protein rearrangement. Here we report the conversion of Cys20 to Ser (C20S protein), which gives the protein the opportunity either to retain the native structure and use the Ser20 O gamma as a ligand or to rearrange and use Cys24. X-ray crystallography demonstrates that the cluster does not use the Ser20 O gamma as a ligand; rather it rearranges to use Cys24. In the C20S protein the [4Fe-4S] cluster has altered stability and redox properties relative to either C20A or the native protein.

Amino Acid Sequence↗

PubMed1995

Steric and conformational features of the aconitase mechanism.

Crystal structures of mitochondrial aconitase with alpha-methylisocitrate and with sulfate bound have been solved and refined at 2.0 A resolution with R factors of 18.2 and 16.8%, respectively. The steric factors and conformational effects observed in both new structures support the proposed mechanism for the overall reaction catalyzed by aconitase. The alternate substrate alpha-methylisocitrate is derived from alpha-methyl-cis-aconitate during crystallization and is observed to bind in the active site in a manner very similar to that observed for isocitrate. The methyl group is accommodated by favorable contact with Ile-425. However, the other potential hydration product of alpha-methyl-cis-aconitate, alpha-methylcitrate, cannot be accommodated in the active site due to steric conflict of the methyl group with Asp-165. The results are consistent with the requirement that cis-aconitate must bind in two ways, in the citrate mode and in the isocitrate mode. Crystals of aconitase with sulfate bound are isomorphous to those with isocitrate bound. However, the structure displays significant conformational changes, providing a model for the substrate-free state of enzyme. Three water molecules bind in place of the C alpha- and C beta-hydroxyl and carboxyl groups of isocitrate, while sulfate binds in place of the C gamma-carboxyl group. Side chains of Ser-642 and Arg-447 in the active site rotate to pair with other side chains in the absence of substrate. The new conformation of Arg-447 triggers a concerted set of shifts which transmits conformational change to the surface of the protein, 30 A from the active site. In the absence of substrate, a chain segment containing the [4Fe-4S] ligand Cys-358 also shifts, resulting in the net translation and reorientation of the Fe-S cluster.

Aconitate Hydratase↗

PubMed1995

Crystal structure and subunit dynamics of the abalone sperm lysin dimer: egg envelopes dissociate dimers, the monomer is the active species.

Lysin is a 16-kD acrosomal protein used by abalone spermatozoa to create a hole in the egg vitelline envelope (VE) by a nonenzymatic mechanism. The crystal structure of the lysin monomer is known at 1.9 A resolution. The surface of the molecule reveals two tracks of basic residues running the length of one surface of the molecule and a patch of solvent-exposed hydrophobic residues on the opposite surface. Here we report that lysin dimerizes via interaction of the hydrophobic patches of monomers. Triton X-100 dissociates the dimer. The crystal structure of the dimer is described at 2.75 A resolution. Fluorescence energy transfer experiments show that the dimer has an approximate KD of 1 microM and that monomers exchange rapidly between dimers. Addition of isolated egg VE dissociates dimers, implicating monomers as the active species in the dissolution reaction. This work represents the first step in the elucidation of the mechanism by which lysin enables abalone spermatozoa to create a hole in the egg envelope during fertilization.

Animals↗

PubMed1994

Diffraction quality crystals of protein X from Azotobacter vinelandii.

Protein X from Azotobacter vinelandii has recently been shown to be either a NADPH oxidase or a NADP+ reductase that interacts specifically with ferredoxin I. Single crystals have been obtained by vapor diffusion from polyethylene glycol 4000 solutions containing 100 mM citrate buffer (pH 5.5). The crystals belong to space group P2(1)2(1)2 with unit cell constants a = 68.9 A, b = 76.9 A, c = 52.8 A and one molecule (M(r) 29,000) per asymmetric unit. The crystals diffract to 2.5 A resolution.

Azotobacter vinelandii↗

PubMed1994

Acid pH crystallization of the basic protein lysin from the spermatozoa of red abalone (Haliotis rufescens).

A new crystal form of dimeric red lysin, a distinctly basic protein (M(r) = 16 070) from the red abalone (Haliotis rufescens), has been obtained using ammonium sulfate as precipitant with a sodium citrate-boric acid-citric acid buffer at pH 4.5. The acid pH crystal form resulted from a study aimed at developing conditions favorable to the sitting-drop vapor-diffusion crystallization of other abalone lysins which do not crystallize at neutral or basic pH conditions. The space group is P222(1) with cell dimensions a = 51.2, b = 47.0, c = 123.8 A and two molecules per asymmetric unit.

Journal Article↗

PubMed1994

Crystal structures of aconitase with trans-aconitate and nitrocitrate bound.

Crystal structures of mitochondrial aconitase with the inhibitors trans-aconitate and nitrocitrate bound to the [4Fe-4S] cluster have been solved and refined at 2.05 A resolution with R-factors of 0.168 and 0.172, respectively. Crystallization of aconitase with the substrates citrate and cis-aconitate has not been possible because the enzyme turns over and selects enzyme with isocitrate bound into the crystal lattice. Therefore we have analyzed crystal structures of the enzyme complexed with inhibitor analogs of these two substrates. The structure with nitrocitrate bound provides a model for citrate binding. The structure with trans-aconitate bound provides a model for cis-aconitate binding in two ways: Fe4 of the [4Fe-4S] cluster is five-coordinate and the carbon at the C beta position is trigonal. These results allow the model for the reaction mechanism to be extended to all three natural substrates of aconitase. The results support a model in which citrate and isocitrate form similar chelate structures related by 180 degrees rotation about the C alpha-C beta bond while the intermediate cis-aconitate binds in either of two ways (citrate mode or isocitrate mode). In both inhibitor complexes a H2O molecule is also bound to Fe4. In the structure with nitrocitrate bound, partial occupancy of sulfate in the active site is observed accompanied by hydroxyl binding to Fe4. Comparison of the structures with isocitrate, trans-aconitate, nitrocitrate and sulfate bound reveals preferred orientations for the three types of oxygens ligated to Fe4 (carboxyl, hydroxyl and H2O) supporting the proposed roles for His101, Asp165 and His167 in the catalytic mechanism.

Aconitate Hydratase↗

PubMed1994

Azotobacter vinelandii ferredoxin I. Alteration of individual surface charges and the [4FE-4S]2+/+ cluster reduction potential.

The structures of Azotobacter vinelandii ferredoxin I (AvFdI) and Peptococcus aerogenes ferredoxin (PaFd), near their analogous [4e-4S]2+/+ clusters, are highly conserved (Backes, G., Mino, Y., Loehr, T.M., Meyer, T.E., Cusanovich, M.A., Sweeney, W.V., Adman, E.T., and Sanders-Loehr, J. (1991) J. Am. Chem. Soc. 11, 2055-2064). Despite these similarities, the reduction potential (E0') of the AvFdI [4Fe-4S]2+/+ cluster is more than 200 mV more negative than that of PaFd. We have tested the contribution that individual amino acid residues make to the control of E0' by converting residues in AvFdI into the corresponding residue in PaFd. Four mutations involved substitutions of negatively charged surface residues with neutral residues and two involved substitution of buried hydrophobic residues. All AvFdI variants were characterized by x-ray crystallography, absorption, CD, EPR, and 1H NMR spectroscopies and by electrochemical methods. For the F25I mutation, significant structural changes occurred that affected the EPR and 1H NMR spectroscopic properties of AvFdI and had a minor influence on E0'. For all other mutations there were no changes in reduction potential. Thus we conclude, that variations in charged surface residues do not account for the observed differences in E0' between the analogous [4Fe-4S]2+/+ cluster of PaFd and AvFdI. These differences are therefore most likely to be due to differences in solvent accessibility.

Amino Acid Sequence↗

PubMed1993

The crystal structure of lysin, a fertilization protein.

Lysin, a protein from abalone sperm, creates a hole in the envelope of the egg, permitting the sperm to pass through the envelope and fuse with the egg. The structure of lysin, refined at 1.9 angstroms resolution, reveals an alpha-helical, amphipathic molecule. The surface of the protein exhibits three features: two tracks of basic residues that span the length of the molecule, a solvent-exposed cluster of aromatic and aliphatic amino acids, and an extended amino-terminal hypervariable domain that is species-specific. The structure suggests possible mechanisms of action.

Amino Acid Sequence↗

PubMed1993

Crystal structures of oxidized and reduced Azotobacter vinelandii ferredoxin at pH 8 and 6.

Crystal structures of Azotobacter vinelandii ferredoxin I (FdI) have been solved and refined at 2.2 to 1.9-A resolution at pH 8 and 6 for both the oxidized and dithionite-reduced proteins. Only the [3Fe-4S] cluster is reduced by dithionite. The four structures (denoted FdI8ox, FdI8red, FdI6ox, and FdI6red) have been compared to address three questions: the effect of reduction at pH 8, the effect of pH change on the structure, and the effect of reduction at pH 6. Comparison of the FdI8ox and FdI8red structures shows that Asp-15 changes conformation in a manner consistent with increased anionic repulsion between this residue and the reduced [3Fe-4S]0 cluster. By revealing an electrostatic interaction between Asp-15 and the [3Fe-4S] cluster, this result supports the conclusion in the accompanying paper (Shen, B., Martin, L. L., Butt, J. N., Armstrong, F. A., Stout, C. D., Jensen, G. M., Stephens, P. J., LaMar, G. N., Gorst, C. M., and Burgess, B. K. (1993) J. Biol. Chem. 268, 25928-25939) that Asp-15 participates in protonation of the reduced [3Fe-4S]0 cluster at acid pH. The [3Fe-4S]0 cluster in the FdI8red structure also displays a distinct shift within the protein as well as internal distortions when compared to the [3Fe-4S]+ cluster in the FdI8ox structure. Comparison of the FdI8ox and FdI6ox structures shows that pH change does not have any significant effect on the [3Fe-4S]+ cluster or surrounding residues. Comparison of the FdI6ox and FdI6red structures shows that reduction at pH 6 also does not have any significant effect on the [3Fe-4S] cluster or Asp-15. The absence of structural change supports the conclusion that at acid pH, the reduced [3Fe-4S] cluster is protonated, i.e. [3Fe-4S]0-H+ (Shen et al., 1993). The cluster is not shifted or distorted as in the FdI8red structure. Instead, the [3Fe-4S]o-H+ cluster FdI8red is structurally similar to the [3Fe-4S]+ cluster (FdI8ox, FdI6ox), which has the same net charge. An Asp-15-Lys-84 salt bridge is observed in all four structures, indicating that Asp-15 is ionized at pH 8 and 6. An ionized state for Asp-15 is also implied by a lack of conformational change in Lys-84; the side chain of this residue rearranges when Asp-15 is substituted with a neutral amino acid (Shen et al., 1993).(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗

PubMed1993

Azotobacter vinelandii ferredoxin I. Aspartate 15 facilitates proton transfer to the reduced [3Fe-4S] cluster.

The [3Fe-4S]+/0 cluster of Azotobacter vinelandii ferredoxin I (AvFdI) has an unusually low and strongly pH-dependent reduction potential (E'0). The reduced cluster exists in two forms, depending upon pH, that exhibit substantially different magnetic circular dichroism (MCD) spectra. Recent studies have established that the MCD changes observed on decreasing the pH from 8.3 (alkaline form) to 6.0 (acid form) cannot be explained either by a change in spin state of the cluster (Stephens, P.J., Jensen, G.M., Devlin, F.J., Morgan, T.V., Stout, C. D., Martin, A.E., and Burgess, B.K. (1991) Biochemistry 30, 3200-3209) or by a major structural change (e.g. ligand exchange) (Stout, C.D. (1993) J. Biol. Chem. 268, 25920-25927). Here, we have examined the influence of aspartate 15 on the pH dependence of the spectroscopic and electrochemical properties of AvFdI by construction of a D15N mutant. Aspartate 15, which is salt-bridged to lysine 84 at the protein surface, is the closest ionizable residue to the [3Fe-4S] cluster. The results show that replacement of aspartate by asparagine results in an approximately 20-mV increase in E'0 for the [3Fe-4S]+/0 cluster at high pH concomitant with an approximately 0.8-pH unit decrease in the pK of the reduced form. The major pH dependence of E'0 is preserved as is the effect observed by MCD. These data eliminate the possibility that the MCD change is due to the presence of Asp-15 and support the conclusion that it originates in direct protonation of the [3Fe-4S]0 cluster, probably on a sulfide ion. Voltammetric studies show that interconversion between [3Fe-4S]+ and [3Fe-4S]0 at acidic pH involves rapid electron transfer followed by proton transfer (for reduction) and then proton transfer followed by electron transfer (for oxidation). Ionized aspartate 15 facilitates proton transfer. Thus, protonation and deprotonation are much slower for D15N relative to the native protein at pH > 5.5. Proton transfer reactions necessary for further reduction of the [3Fe-4S]0 cluster to the [3Fe-4S]- and [3Fe-4S]2- states are also retarded in D15N. The results suggest that the carboxylate-ammonium salt bridge afforded by Asp-15-Lys-84 conducts protons between the cluster and solvent H2O molecules. Overproduction of D15N FdI, but not native FdI, in A. vinelandii has a negative effect on the growth rate of the organism, suggesting that the rate of protonation or deprotonation of the [3Fe-4S]0 cluster may be important in vivo.

Amino Acid Sequence↗