Redox properties of several bacterial ferredoxins using square wave voltammetry

The equilibrium reduction potential of the 2[4Fe-4S] ferredoxin (Fd) isolated from four different bacterial strains was determined at a methyl viologen-modified gold electrode using square wave voltammetry. The observed reduction potential at pH 8 for Clostridium thermoaceticum Fd was -385 mV; Clostridium pasteurianum, -393 mV; Clostridium thermosaccharolyticum, -408 mV; and Chromatium vinosum, -460 mV versus normal hydrogen electrode at 25 degrees C. The reduction potential of the C. pasteurianum Fd was found to be pH independent from pH 6.4 to 8.7, indicating that the electron transfer mechanism does not involve proton exchange. In contrast, the reduction potential of the C. thermosaccharolyticum Fd was found to be pH dependent from pH 6.4 to 8.7, with pKox approximately 7 and pKred approximately 7.5. The +30 mV change in reduction potential from pH 8.7 to 6.4 was attributed to an electrostatic interaction between the iron-sulfur cluster II and the protonated histidine 2 residue located about 6 A away. The Ch. vinosum Fd interacted reversibly at the methyl viologen-modified gold electrode, and its reduction potential was verified using visible spectroelectrochemistry. The reduction potential of Ch. vinosum Fd was found to be 30 mV more positive than previously reported. The similarities of the bacterial Fd reduction potentials are discussed in terms of the homology of their primary structure as reflected by the similarities in the visible and circular dichroic spectra.     

Spectroscopic and functional properties of novel 2[4Fe-4S] cluster-containing ferredoxins from the green sulfur bacterium Chlorobium tepidum

Two distinct ferredoxins, Fd I and Fd II, were isolated and purified to homogeneity from photoautotrophically grown Chlorobium tepidum, a moderately thermophilic green sulfur bacterium that assimilates carbon dioxide by the reductive tricarboxylic acid cycle. Both ferredoxins serve a crucial role as electron donors for reductive carboxylation, catalyzed by a key enzyme of this pathway, pyruvate synthase/pyruvate ferredoxin oxidoreductase. The reduction potentials of Fd I and Fd II were determined by cyclic voltammetry to be -514 and -584 mV, respectively, which are more electronegative than any previously studied Fds in which two [4Fe-4S] clusters display a single transition. Further spectroscopic studies indicated that the CD spectrum of oxidized Fd I closely resembled that of Fd II; however, both spectra appeared to be unique relative to ferredoxins studied previously. Double integration of the EPR signal of the two Fds yielded approximately approximately 2.0 spins per molecule, compatible with the idea that C. tepidum Fd I and Fd II accept 2 electrons upon reduction. These results suggest that the C. tepidum Fd I and Fd II polypeptides each contain two bound [4Fe-4S] clusters. C. tepidum Fd I and Fd II are novel 2[4Fe-4S] Fds, which were shown previously to function as biological electron donors or acceptors for C. tepidum pyruvate synthase/pyruvate ferredoxin oxidoreductase (Yoon, K.-S., Hille, R., Hemann, C. F., and Tabita, F. R. (1999) J. Biol. Chem. 274, 29772-29778). Kinetic measurements indicated that Fd I had approximately 2.3-fold higher affinity than Fd II. The results of amino acid sequence alignments, molecular modeling, oxidation-reduction potentials, and spectral properties strongly indicate that the C. tepidum Fds are chimeras of both clostridial-type and chromatium-type Fds, suggesting that the two Fds are likely intermediates in the evolutional development of 2[4Fe-4S] clusters compared with the well described clostridial and chromatium types.     

Characterization of the recombinant Clostridium pasteurianum ferredoxin and comparison of its properties with those of the native protein

Ferredoxins (Fds) constitute an important class of nonheme iron-sulfur proteins. One of the most studied Fds is the [8Fe-8S] Fd from Clostridium pasteurianum. The gene for this Fd has previously been cloned and sequenced. We report the expression of this Fd in Escherichia coli, and the characterization and comparison of this recombinant protein to the native Fd. We have found that the purified recombinant protein has the same enzymatic, redox, magnetic and electronic properties as the native Fd isolated from C. pasteurianum, which indicates that the two [4Fe-4S] clusters present in the Fd were correctly formed in E. coli.     

Oxidation-reduction properties of several low potential iron-sulfur proteins and of methylviologen.

Apparent oxidation-reduction potentials at pH 7.0 and 25 degrees C were determined using the H2-hydrogenase system with ferredoxins from the following sources: Clostridium pasteurianum, -403 mV; C tartarovorum, -424 mV; C. acidi-urici, -434 mV; Peptococcus aerogenes, -427 mV; Chromatium D, -482 mV (pH 8.0); B. polymyxa, Fd I, -377 mV, and Fd II, -422 mV; and spinach, -428 mV. The pH dependence of these values was variable, ranging from -2 to -24 mV/pH unit increase for different ferredoxins. Over the range of buffer concentrations between 0.05 and 0.2 M, the potentials did not vary significantly. The number of electrons transferred during reduction (as determined by integrations of EPR spectra and by dithionite titration) is 2 for the first five proteins, while potentiometric data for all the cases fit a Nernst equation for which n = 1. The E degrees' value for the redox indicator methylviologen at pH 7.4 was found to be -460 mV, according to both the H2-hydrogenase system and cyclic voltammetry, significantly different from the value previously reported at higher pH's. Additionally, the presence of C. pasteuranum ferredoxin appears to shift the E degrees value of methylviologen to even more negative values. An analysis of sources of error inherent with potential determinations with H2 and hydrogenase is presented. The electronic and EPR spectra of P. aerogenes ferredoxin, for which the x-ray structure has been published, are given here. It appears that the determination of potentials of ferredoxin and other low-potential porteins with the H2-hydrogenase system affords certain experimental advantages over alternative methods currently employed with these and similar substances.     

Design and functional expression in Escherichia coli of a synthetic gene encoding Clostridium pasteurianum 2[4Fe-4S] ferredoxin.

A gene encoding the exact sequence of Clostridium pasteurianum 2[4Fe-4S] ferredoxin and containing 11 unique restriction endonuclease cleavage sites has been synthesized and cloned in Escherichia coli. The synthetic gene is efficiently expressed in E. coli and its product has been purified and characterized. The N-terminal sequence is identical to that of the protein isolated from C. pasteurianum and the recombinant ferredoxin contains the exact amount of [4Fe-4S] clusters (2 per monomer) expected for homogeneous holoferredoxin. It displays reduction potential and kinetic parameters as electron donor to C. pasteurianum hydrogenase I identical to those determined for the native ferredoxin. All of these properties demonstrate that the 2[4Fe-4S] ferredoxin expressed in E. coli is identical to the parent clostridial protein.     

Characteristic features of the heterologous functional synthesis in Escherichia coli of a 2[4Fe-4S] ferredoxin

Different strategies have been used to express synthetic genes all encoding Clostridium pasteurianum 2[4Fe-4S] ferredoxin (Fd) in Escherichia coli. The polypeptide can be produced as the C-terminal addition to a hybrid Cro::Protein A fusion protein lacking the metallic centers. The incorporation of the [4Fe-4S] clusters into the cleaved apoFd cannot be carried out in the same conditions as those affording holoFd from purified C. pasteurianum apoFd. In contrast, fully functional Fds can be produced from non-fused synthetic genes under the dependence of strong promoters. The yields of recombinant Fd, although sufficient to purify significant quantities of protein, are limited by the very short half-life of the 2[4Fe-4S] Fd in E. coli, irrespective of the expression system used. These features are characteristic of 2[4Fe-4S] Fds when compared with the far more stable recombinant rubredoxin, and probably other small iron-sulfur proteins which have already been produced in high yields. The reasons for the high turnover of 2[4Fe-4S] Fds are discussed.     

Electron transfer by ferredoxin:NADP+ reductase. Rapid-reaction evidence for participation of a ternary complex

Rapid reaction studies presented herein show that ferredoxin:NADP+ oxidoreductase (FNR, EC 1.18.1.2) catalyzes electron transfer from spinach ferredoxin (Fd) to NADP+ via a ternary complex, Fd X FNR X NADP+. In the absence of NADP+, reduction of ferredoxin:NADP+ reductase by Fd was much slower than the catalytic rate: 37-80 s-1 versus at least 445 e-s-1; dissociation of oxidized spinach ferredoxin (Fdox) from one-electron reduced ferredoxin:NADP+ reductase (FNRsq) limited the reduction of FNR. This confirms the steady-state kinetic analysis of Masaki et al. (Masaki, R., Yoshikaya, S., and Matsubara, H. (1982) Biochim. Biophys. Acta 700, 101-109). Occupation of the NADP+ binding site of FNR by NADP+ or by 2',5'-ADP (a nonreducible NADP+ analogue) greatly increased the rate of electron transfer from Fd to FNR, releiving inhibition by Fdox. NADP+ (and 2',5'-ADP) probably facilitate the dissociation of Fdox; equilibrium studies have shown that nucleotide binding decreases the association of Fd with FNR (Batie, C. J. (1983) Ph.D. dissertation, Duke University; Batie, C. J., and Kamin, H. (1982) in Flavins and Flavoproteins VII (Massey, V., and Williams, C. H., Jr., eds) pp. 679-683, Elsevier, New York; Batie, C.J., and Kamin, H. (1982) Fed. Proc. 41, 888; and Batie, C.J., and Kamin, H. (1984) J. Biol. Chem. 259, 8832-8839). Premixing Fd with FNR was found to inhibit the reaction of the flavoprotein with NADP+ and with NADPH; thus, substrate binding may be ordered, NADP+ first, then Fd. FNRred and NADP+ very rapidly formed an FNRred X NADP+ complex with flavin to nicotinamide charge transfer bands. The Fdred X NADP+ complex then relaxed to an equilibrium species; the spectrum indicated a predominance of FNRox X NADPH charge-transfer complex. However, charge-transfer species were not observed during turnover; thus, their participation in catalysis of electron transfer from Fd to NADP+ remains uncertain. The catalytic rate of Fd to NADP+ electron transfer, as well as the rates of electron transfer from Fd to FNR, and from FNR to NADP+ were decreased when the reactants were in D2O; diaphorase activity was unaffected by solvent. On the basis of the data presented, a scheme for the catalytic mechanism of catalysis by FNR is presented.     

Secondary structure extensions in Pyrococcus furiosus ferredoxin destabilize the disulfide bond relative to that in other hyperthermostable ferredoxins. Global consequences for the disulfide orientational heterogeneity.

The single cubane cluster ferredoxin (Fd) from the hyperthermophilic archaeon Pyrococcus furiosus (Pf) possesses several unique properties when compared even to Fds from other hyperthermophilic archaea or bacteria. These include an equilibrium molecular heterogeneity, a six- to seven-residue increase in size, an Asp rather than the Cys as one cluster ligand, and a readily reducible disulfide bond. NMR assignments and determination of both secondary structure and tertiary contacts remote from the paramagnetic oxidized cluster of Pf 3Fe Fd with an intact disulfide bond reported previously (Teng Q., Zhou, Z. H., Smith, E. T., Busse, S. C., Howard, J. B. Adams, M. W. W., and La Mar, G. (1994) Biochemistry 33, 6316-6328) are extended here to the 4Fe oxidized cluster WT (1H and 15N) and D14C (1H only) Fds with an intact disulfide bond and to the 4Fe oxidized WT Fd (1H and 15N) with a cleaved disulfide bond. All forms are shown to possess a long (13-member) alpha-helix, two beta-sheets (one double-, one triple-stranded), and three turns outside the cluster vicinity, each with tertiary contacts among themselves as found in other Fds. While the same secondary structural elements, with similar tertiary contacts, are found in other hyperthermostable Fds, Pf Fd has two elements, the long helix and the triple-stranded beta-sheet, that exhibit extensions and form multiple tertiary contacts. All Pf Fd forms with an intact disulfide bond exhibit a dynamic equilibrium heterogeneity which is shown to modulate a hydrogen-bonding network in the hydrophobic core that radiates from the Cys21-Cys48 disulfide bond and encompasses residues Lys36, Val24, Cys21, and Cys17 and the majority of the long helix. The heterogeneity is attributed to population of the alternate S and R chiralities of the disulfide bond, each destabilized by steric interactions with the extended alpha-helix. Comparison of the chemical shifts and their temperature gradients reveals that the molecular structure of the protein with the less stable R disulfide resembles that of the Fd with a cleaved disulfide bond. Both cluster architecture (3Fe vs 4Fe) and ligand mutation (Cys for Asp14) leave the disulfide orientational heterogeneity largely unperturbed. It is concluded that the six- to seven-residue extension that results in a longer helix and larger beta-sheet in Pf Fd, relative to other hyperthermostable Fds, more likely serves to destabilize the disulfide bond, and hence make it more readily reducible, than to significantly increase protein thermostability.     

Characterization of the selenium-substituted 2 [4Fe-4Se] ferredoxin from Clostridium pasteurianum

The sulfur atoms of the two [4Fe-4S] clusters present in the ferredoxin from Clostridium pasteurianum have been replaced by selenium. The substitution is readily carried out by incubating the apoferredoxin with excess amounts of Fe3+, selenite, and dithiothreitol under anaerobic conditions. The UV-visible absorption spectrum of the Se-substituted ferredoxin, the core extrusion of its active sites, and analyses of its iron and selenium contents show that it contains two [4Fe-4Se] clusters. The Se-substituted ferredoxin is considerably less resistant to oxygen or to acidic and alkaline pH than the native ferredoxin: the half-lives of the former are 20-500 times shorter than those of the latter. The native ferredoxin and the Se-substituted ferredoxin display similar kinetic properties when used as electron donors to the hydrogenase from C. pasteurianum. It is of note, however, that the Km and Vmax values are lower for the 2[4Fe-4Se] ferredoxin than for the 2[4Fe-4S] ferredoxin. Reductive and oxidative titrations with dithionite and with thionine, respectively, show that both ferredoxins are two-electron carriers. The redox potentials of the ferredoxins have been measured by equilibrating them with the H2/H+ couple via hydrogenase: values of -423 and -417 mV have been found for the 2[4Fe-4S] ferredoxin and 2[4Fe-4Se] ferredoxin, respectively. Ferredoxins containing both chalcogenides in their [4Fe-4X] (X = S, Se) clusters have been prepared by reconstitution reactions involving mixtures of sulfide and selenide: the latter experiments show that sulfide and selenide are equally reactive in the incorporation of [4Fe-4X] (X = S, Se) sites into ferredoxin. The present report, together with former studies, establishes the general feasibility of the Se/S substitution in [2Fe-2S] and in [4Fe-4S] clusters of proteins and of synthetic analogues.     

Cloning and characterization of ferredoxin and ferredoxin-NADP+ reductase from human malaria parasite

The human malaria parasite (Plasmodium falciparum) possesses a plastid-derived organelle called the apicoplast, which is believed to employ metabolisms crucial for the parasite's survival. We cloned and studied the biochemical properties of plant-type ferredoxin (Fd) and Fd-NADP+ reductase (FNR), a redox system that potentially supplies reducing power to Fd-dependent metabolic pathways in malaria parasite apicoplasts. The recombinant P. falciparum Fd and FNR proteins were produced by synthetic genes with altered codon usages preferred in Escherichia coli. The redox potential of the Fd was shown to be considerably more positive than those of leaf-type and root-type Fds from plants, which is favourable for a presumed direction of electron flow from catabolically generated NADPH to Fd in the apicoplast. The backbone structure of P. falciparum Fd, as solved by X-ray crystallography, closely resembles those of Fds from plants, and the surface-charge distribution shows several acidic regions in common with plant Fds and some basic regions unique to this Fd. P. falciparum FNR was able to transfer electrons selectively to P. falciparum Fd in a reconstituted system of NADPH-dependent cytochrome c reduction. These results indicate that an NADPH-FNR-Fd cascade is operative in the apicoplast of human malaria parasites.     

Cyanidioschyzon merolae ferredoxin: a high resolution crystal structure analysis and its thermal stability

Cyanidioschyzon merolae (Cm) is a single-cell red alga that grows under moderately thermophilic (40-50°C), acidic (pH 1-3) conditions. We purified a Cm ferredoxin (Fd) that was characterized as a plant-type [2Fe-2S] Fd by physicochemical techniques. X-ray crystallography revealed that the overall three-dimensional structure of CmFd was highly similar to, but slightly different from, the [2Fe-2S] Fd from Spinacia oleracea, whose growth temperature is 15-20°C. Therefore, slight structural differences, including non-covalent-bond number and amino acid sequence, may underlie the differential thermostabilities of the plant-type Fds.     

Rubredoxin from Clostridium perfringens: complete amino acid sequence and participation in nitrate reduction

The complete primary structure of rubredoxin (Rd) isolated from Clostridium perfringens was sequenced to be: MKKFICDVCGYIYDPAVGDPDNGVEPGTEFKDIPDDWVCPLCGVDKSQFSETEE. The sequence was highly homologous to that of C. pasteurianum Rd but was different at 13 sites out of the total 54 amino acid residues (76% homology). It contained 1 Fe atom, 4 cysteine residues, and no labile sulfur, had a molecular weight of 6,056, and shared the general properties of classical anaerobic Rds. The pI was 4.4. The Rd was reduced with NADH in the presence of a specific NAD(P)H oxidoreductase preparation from the bacterium. The Km value of nitrate reductase for Rd as an electron-donor was 12 microM, a value comparable to that of the 13 microM for ferredoxin (Fd). These results taken together provide additional support for its role as the electron carrier in the nitrate reductase system [Seki, S., Ikeda, A., and Ishimoto, M. (1988) J. Biochem. 103, 583-584].     

Study of the influence of NH ⃛S hydrogen bonds on the reduction potential in Clostridium pasteurianum 2(4Fe-4S) ferredoxin using deuterium exchange

The effect of deuterium substitution of exchangeable hydrogen atoms on the reduction potential of Clostridium pasteurianum 2(4Fe-4S) ferredoxin has been studied. The studies were conducted to determine if NH ?S hydrogen bonds to the iron–sulfur cluster are dominant in the mechanism of influence of the protein on cluster reduction potential, as has been proposed [Carter, C. W. (1977) J. Biol. Chem. 252 , 7802–7811]. Deuteration of the slowly exchangeable hydrogen atoms, however, yields essentially no shift in the reduction potential (?0.2 ± 0.8 mV), suggesting that NH ?S bonds are not important modifiers of cluster reduction potential in this protein.     

Oxidation-reduction properties of the two Fe4S4 clusters in Clostridium pasteurianum ferredoxin

The ferredoxin from Clostridium pasteurianum contains two Fe4S4 clusters. In this paper we determine their oxidation-reduction midpoint potentials; we find them to be essentially identical (within 10 mV) and to have pH-independent Em values of -412 +/- 11 mV from pH 6.3 to 10.0.     

FdC1, a novel ferredoxin protein capable of alternative electron partitioning, increases in conditions of acceptor limitation at photosystem I

In higher plants, [2Fe-2S] ferredoxin (Fd) proteins are the unique electron acceptors from photosystem I (PSI). Fds are soluble, and distribute electrons to many enzymes, including Fd:NADP(H) reductase (FNR), for the photoreduction of NADP(+). In addition to well studied [2Fe-2S] Fd proteins, higher plants also possess genes for significantly different, as yet uncharacterized Fd proteins, with extended C termini (FdCs). Whether these FdC proteins function as photosynthetic electron transfer proteins is not known. We examined whether these proteins play a role as alternative electron acceptors at PSI, using quantitative RT-PCR to follow how their expression changes in response to acceptor limitation at PSI, in mutant Arabidopsis plants lacking 90-95% of photosynthetic [2Fe-2S] Fd. Expression of the gene encoding one FdC protein, FdC1, was identified as being strongly up-regulated. We confirmed that this protein was chloroplast localized and increased in abundance on PSI acceptor limitation. We purified the recombinant FdC1 protein, which exhibited a UV-visible spectrum consistent with a [2Fe-2S] cluster, confirmed by EPR analysis. Measurements of electron transfer show that FdC1 is capable of accepting electrons from PSI, but cannot support photoreduction of NADP(+). Whereas FdC1 was capable of electron transfer with FNR, redox potentiometry showed that it had a more positive redox potential than photosynthetic Fds by around 220 mV. These results indicate that FdC1 electron donation to FNR is prevented because it is thermodynamically unfavorable. Based on our data, we speculate that FdC1 has a specific function in conditions of acceptor limitation at PSI, and channels electrons away from NADP(+) photoreduction.     

A post genomic characterization of Arabidopsis ferredoxins

In higher plant plastids, ferredoxin (Fd) is the unique soluble electron carrier protein located in the stroma. Consequently, a wide variety of essential metabolic and signaling processes depend upon reduction by Fd. The currently available plant genomes of Arabidopsis and rice (Oryza sativa) contain several genes encoding putative Fds, although little is known about the proteins themselves. To establish whether this variety represents redundancy or specialized function, we have recombinantly expressed and purified the four conventional [2Fe-2S] Fd proteins encoded in the Arabidopsis genome and analyzed their physical and functional properties. Two proteins are leaf type Fds, having relatively low redox potentials and supporting a higher photosynthetic activity. One protein is a root type Fd, being more efficiently reduced under nonphotosynthetic conditions and supporting a higher activity of sulfite reduction. A further Fd has a remarkably positive redox potential and so, although redox active, is limited in redox partners to which it can donate electrons. Immunological analysis indicates that all four proteins are expressed in mature leaves. This holistic view demonstrates how varied and essential soluble electron transfer functions in higher plants are fulfilled through a diversity of Fd proteins.     

Hyperproduction of recombinant ferredoxins in escherichia coli by coexpression of the ORF1-ORF2-iscS-iscU-iscA-hscB-hs cA-fdx-ORF3 gene cluster.

Fe-S proteins acquire Fe-S clusters by an unknown post-translational mechanism. To study the in vivo synthesis of the Fe-S clusters, we constructed an experimental system to monitor the expressed ferredoxin (Fd) as a reporter of protein-bound Fe-S clusters assembled in Escherichia coli. Overexpression of five Fds in a T7 polymerase-based system led to the formation of soluble apoFds and mature holoFds, indicating that assembly of the Fe-S cluster into apoFd polypeptides is a rate-limiting step. We examined the coexpression of the E. coli ORF1-ORF2-iscS-iscU-iscA-hscB-hsc A-fdx-ORF3 gene cluster, which has recently been suggested to be involved in the formation or repair of Fe-S protein [Zheng, L., Cash, V.L., Flint, D.H., and Dean, D.R. (1998) J. Biol. Chem. 273, 13264-13272], with reporter Fds using compatible plasmids. The production of all five reporter holoFds examined was dramatically increased by the coexpression of the gene cluster, and apparent specificity to the polypeptides or to the type of Fe-S clusters was not observed. The increase in holoFd production was observed under the coexpression conditions in all culture media examined, with either 2 x YT medium or Terrific broth, and with or without supplemental cysteine or iron. These results indicate that the proteins encoded by the gene cluster are involved in the assembly of the Fe-S clusters in a wide variety of Fe-S proteins.     

Apparent oxidation-reduction potential of Clostridium acidi-urici ferredoxin. Effect of pH, ionic strength, and amino acid replacements.

The effects of pH and ionic strength on the midpoint reduction potential (Emp) of Clostridium acidi-urici ferredoxin were determined using hydrogen gas and hydrogenase. The Emp of native ferredoxin at 24-25 degrees in 0.1 M Tris-chloride buffer, pH 7.0, is--0.434 V. In the pH range examined, the Emp becomes approximately 13 mv more negative per each pH unit increase. A plot of the log of ionic strength versus the apparent Emp of ferredoxin in 0.1 M Tris-chloride buffer, pH 7.5, Was linear over the range of 1.0 to 0.01 ionic strength with Emp values of--0.414 and--0.475 V, respectively, at these extremes. This effect is the same with sodium chloride, sodium bromide, or ammonium sulfate. Potassium phosphate buffer caused a similar change, but the absolute values of Emp differed from those obtained in the presence of the other salts. This effect of pH and ionic strength on Emp may be general for clostridial-type (Fe4S4)2-ferredoxins, since the apparent Emp of Clostridium pasteurianum ferredoxin is affected in a similar manner by these two variables. The Emp of this ferredoxin in 0.1 M Tris-chloride buffer pH 7.0, is--0.405 V. Since the NH2-terminal amino acid residue, Ala1, and Tyr2 of C. acidi urici ferredoxin are near an (Fe4S4)2-cluster in the protein, the apparent Emp of derivatives that contained amino acid replacements in these two positions were determined. Under similar conditions, the Emp of most of the 13 derivatives examined, including those of [Leu2]- and[3-NH2-Tyr30]ferredoxin, is approximately the same as that of native ferredoxin. However, the Emp of [His2]ferredoxin is approximately 15 mv more positive, whereas that of [Trp2]ferredoxin is 22 mv more negative than that of native C. acidi-urici ferredoxin. Variations in sodium chloride concentration and pH also affected the apparent Emp of the derivatives. It is suggested that the changes observed in the Emp of C. acidi-urici ferredoxin are caused by protein conformational changes.     

Two-dimensional NMR investigation of iron-sulfur cluster electronic and molecular structure of oxidized Clostridium pasteurianum ferredoxin. Interpretability of contact shifts in terms of cysteine orientation.

A two-dimensional NMR study has been carried out on the four-iron clusters of a bacterial oxidized ferredoxin for the purpose of investigating the relationship between contact shift patterns and the orientation of the individual coordinated cysteines. The ferredoxin from Clostridium pasteurianum, CpFdox, was selected because of its extensive sequence homology, and likely close structural similarity, to the crystallographically characterized ferredoxin from Peptococcus aerogenes, Pa Fdox (Adman, E.T., Sieker, L.C., and Jensen, L. H. (1973) J. Biol. Chem. 248, 3987-3996). Rapid data collection rates with minimal but adequate acquisition time allowed the detection of numerous CpFdox cross-peaks from the contact-shifted and strongly relaxed coordinated cysteinyl C beta H protons in the resolved 10-20 ppm window. Relatively strong magnitude COSY cross peaks from the resolved eight cysteinyl C beta H resonance unambiguously locate the geminal C beta H partner for each residue; weaker cross-peaks locate the C alpha Hs from three of the residues. The geminal nature of the magnitude-COSY detected partners to the resolved C beta H peaks is confirmed by strong NOESY cross-peaks. The NOESY spectra, moreover, assign an additional two cysteinyl C alpha H resonances. The present results confirm some previous one-dimensional NOE assignments, revise others, and locate resonances previously undetected (Bertini, I., Briganti, F., Luchinat, C., and Scozzafara, A. (1990) Inorg. Chem. 29, 1874-1880). A striking pairwise pseudo-symmetry in cysteinyl contact shift patterns is observed which is attributed to the previously recognized pseudo-symmetry in the crystal of PaFdox. A detailed analysis of the structural/electronic determinants of the coordinated cysteine C beta H contact shift pattern is made, and the NMR data necessary for unique interpretation are identified. It is shown that analysis of the relaxation properties of cysteine beta-methylene protons provides the stereospecific assignments necessary for comparison of shift ratios with crystallographic structural data. The available structural data on PaFdox (Backes, G., Mino, Y., Loehr, T., Meyer, T., Cusanovich, M., Sweeney, W., Adman, E., and Sanders-Loehr, J. (1991) J. Am. Chem. Soc. 13, 2055-2064) are qualitatively but not quantitatively consistent with the observed cysteinyl contact shift pattern, with the NMR data reflecting more asymmetry than previous studies. A tentative assignment of a single pair of symmetry-related cysteines is proposed.(ABSTRACT TRUNCATED AT 400 WORDS)