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• W2091880825 abstract "The 2[4Fe-4S] ferredoxin from Chromatium vinosum arises as one prominent member of a recently defined family of proteins found in very diverse bacteria. The potentiometric circular dichroism titrations of the protein and of several molecular variants generated by site-directed mutagenesis have established that the reduction potentials of the two clusters differ widely by almost 200 mV. This large difference has been confirmed by electrochemical methods, and each redox transition has been assigned to one of the clusters. The unusually low potential center is surprisingly the one that displays a conventional CX1 X2CX3 X4C (Xn, variable amino acid) binding motif and a structural environment similar to that of clusters having less negative potentials. A comparison with other ferredoxins has highlighted factors contributing to the reduction potential of [4Fe-4S] clusters in proteins. (i) The loop between the coordinating cysteines 40 and 49 and the C terminus α-helix of C. vinosum ferredoxin cause a negative, but relatively moderate, shift of ∼60 mV for the nearby cluster. (ii) Very negative potentials, below −600 mV, correlate with the presence of a bulky side chain in positionX4 of the coordinating triad of cysteines. These findings set the framework in which previous observations on ferredoxins can be better understood. They also shed light onto the possible occurrence and properties of very low potential [4Fe-4S] clusters in less well characterized proteins. The 2[4Fe-4S] ferredoxin from Chromatium vinosum arises as one prominent member of a recently defined family of proteins found in very diverse bacteria. The potentiometric circular dichroism titrations of the protein and of several molecular variants generated by site-directed mutagenesis have established that the reduction potentials of the two clusters differ widely by almost 200 mV. This large difference has been confirmed by electrochemical methods, and each redox transition has been assigned to one of the clusters. The unusually low potential center is surprisingly the one that displays a conventional CX1 X2CX3 X4C (Xn, variable amino acid) binding motif and a structural environment similar to that of clusters having less negative potentials. A comparison with other ferredoxins has highlighted factors contributing to the reduction potential of [4Fe-4S] clusters in proteins. (i) The loop between the coordinating cysteines 40 and 49 and the C terminus α-helix of C. vinosum ferredoxin cause a negative, but relatively moderate, shift of ∼60 mV for the nearby cluster. (ii) Very negative potentials, below −600 mV, correlate with the presence of a bulky side chain in positionX4 of the coordinating triad of cysteines. These findings set the framework in which previous observations on ferredoxins can be better understood. They also shed light onto the possible occurrence and properties of very low potential [4Fe-4S] clusters in less well characterized proteins. [4Fe-4S] clusters are present in a wide variety of proteins; they are often coordinated to amino acids organized in characteristic motifs (1Matsubara H. Saeki K. Adv. Inorg. Chem. 1992; 38: 223-280Crossref Scopus (173) Google Scholar) corresponding to protein domains, sometimes identified by x-ray crystallography (see Ref. 2Dauter Z. Wilson K.S. Sieker L.C. Meyer J. Moulis J.-M. Biochemistry. 1997; 36: 16065-16073Crossref PubMed Scopus (127) Google Scholar and references therein). A pair of [4Fe-4S] clusters is found in many bacterial ferredoxins (3Otaka E. Ooi T. J. Mol. Evol. 1987; 26: 257-267Crossref PubMed Scopus (43) Google Scholar) and other electron transfer proteins and enzymes (see Fig. 1 in Ref. 4Quinkal I. Davasse V. Gaillard J. Moulis J.-M. Protein Eng. 1994; 7: 681-687Crossref PubMed Scopus (48) Google Scholar). In most cases these clusters are bound to the proteins through a pair of CXXCXXC … CP (X, variable amino acid) motifs containing all necessary cysteine ligands. Extensive work on the simplest ferredoxins over the last 35 years has established that the two clusters are relatively close, at a distance of approximately 10 Å thus enabling them to interact magnetically, and that they display similar reduction potentials, generally around −400 ± 100 mV (normal hydrogen electrode). Until now, these generic properties have not been conclusively challenged in any fully characterized molecule containing such 2[4Fe-4S] domains. Proteins with homologous coordinating motifs are known in which one of the [4Fe-4S] clusters is substituted by a [3Fe-4S] center, often as a result of the loss of one of the cysteine ligands (5Stout G.H. Turley S. Sieker L.C. Jensen L.H. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 1020-1022Crossref PubMed Scopus (103) Google Scholar). Consequently, these proteins exhibit properties that are clearly different from those of molecules containing two [4Fe-4S] centers (6Beinert H. Thomson A.J. Arch. Biochem. Biophys. 1983; 222: 333-361Crossref PubMed Scopus (196) Google Scholar).Recently, the 2[4Fe-4S] ferredoxin from the purple sulfur photosynthetic bacterium Chromatium vinosum has been further investigated (7Huber J.G. Gaillard J. Moulis J.-M. Biochemistry. 1995; 34: 194-205Crossref PubMed Scopus (45) Google Scholar), following its initial characterization (8Bachofen R. Arnon D.I. Biochim. Biophys. Acta. 1966; 120: 259-265Crossref PubMed Scopus (38) Google Scholar), in an effort to assess the influence of specific sequence elements (including a 6-amino acid insertion between two cysteine ligands and a long C-terminal extension) on the properties of the [4Fe-4S] clusters. It was found that, in contrast to other 2[4Fe-4S] ferredoxins without these elements, the electronic communication between the two clusters was apparently impaired (7Huber J.G. Gaillard J. Moulis J.-M. Biochemistry. 1995; 34: 194-205Crossref PubMed Scopus (45) Google Scholar). The gene encoding C. vinosumferredoxin (CvFd) 1The abbreviations used are: CvFd,Chromatium vinosum ferredoxin; Cp, Clostridium pasteurianum. 1The abbreviations used are: CvFd,Chromatium vinosum ferredoxin; Cp, Clostridium pasteurianum. was later cloned and expressed in Escherichia coli (9Moulis J.-M. Biochim. Biophys. Acta. 1996; 1308: 12-14Crossref PubMed Scopus (18) Google Scholar), and the structure of the protein was determined (10Moulis J.-M. Sieker L.C. Wilson K.S. Dauter Z. Protein Sci. 1996; 5: 1765-1775Crossref PubMed Scopus (63) Google Scholar). These studies provide the necessary background for exploring the structure-function relationships in CvFd by protein engineering, and this report presents our initial efforts toward this aim.The properties of Fe-S clusters can be probed by a number of spectroscopic methods. The absorption spectra are generally broad as they result from overlapping charge transfer bands in the visible-near UV range. The resolution of these spectra can be significantly improved by the implementation of circular dichroism (CD) which provides both a characteristic pattern for each cluster type and a sensitive monitor of changes affecting either the cluster or the protein (11Stephens P.J. Thomson A.J. Dunn J.B.R. Keiderling T.A. Rawlings J. Rao K.K. Hall D.O. Biochemistry. 1978; 17: 4770-4778Crossref PubMed Scopus (98) Google Scholar). The use of the method as an accurate analytical tool is broadened when the CD spectra of electron transfer proteins can be monitored as a function of the applied potential (12Link T.A. Hatzfeld O.M. Unalkat P. Shergill J.K. Cammack R. Mason J.R. Biochemistry. 1996; 35: 7546-7552Crossref PubMed Scopus (58) Google Scholar). Indeed, both a measure of the reduction potential and an estimate of the structural changes induced by the addition or removal of electrons are then provided.The results of such studies, together with direct electrochemical measurements, are reported herein for CvFd and a series of molecular variants. The comparison with the well characterized 2[4Fe-4S] ferredoxin from Clostridium pasteurianum (CpFd) reveals that the [4Fe-4S] clusters in CvFd display unusual redox properties which help explain previously observed differences between these proteins (7Huber J.G. Gaillard J. Moulis J.-M. Biochemistry. 1995; 34: 194-205Crossref PubMed Scopus (45) Google Scholar). These data also provide unprecedented compelling evidence that the protein domains containing two [4Fe-4S] clusters found in widely different biological systems do not always display similar physicochemical and functional properties.RESULTSMolecular Variants of CvFdFig. 1 highlights the targets for site-directed mutagenesis investigated in CvFd. The first set of variants involves modifications of the protein environment around cluster II which is coordinated by Cys-18, Cys-37, Cys-40, and Cys-49. In the variant referred to as Δ1 the loop (residues 41–48) between two of the cysteine ligands of cluster II (10Moulis J.-M. Sieker L.C. Wilson K.S. Dauter Z. Protein Sci. 1996; 5: 1765-1775Crossref PubMed Scopus (63) Google Scholar) has been replaced by two residues (Ala-Gln), therefore converting the sequence to a more conventional binding motif (CXXCAQC … CP) for [4Fe-4S] clusters. In the K74− variant, half (residues 74–82) of the C-terminal α-helix has been removed. In Δ1K74− the two modifications were combined, and in K74E the buried hydrophilic side chain of lysine (10Moulis J.-M. Sieker L.C. Wilson K.S. Dauter Z. Protein Sci. 1996; 5: 1765-1775Crossref PubMed Scopus (63) Google Scholar) has been substituted by glutamic acid. In two more variants, Y44C and Y44S, residue Tyr-44 of loop Δ1 has been replaced by a cysteine or a serine.The second set of variants, namely D12G, V13G, and Y30F, contains single site replacements introduced close to cluster I which is coordinated by Cys-8, Cys-11, Cys-14, and Cys-53.Circular Dichroism SpectroscopySince CvFd apparently deviates from other 2[4Fe-4S] ferredoxins in its ability to convey intramolecular electron transfer between its clusters (7Huber J.G. Gaillard J. Moulis J.-M. Biochemistry. 1995; 34: 194-205Crossref PubMed Scopus (45) Google Scholar), evidence for unusual spectroscopic properties was sought using CD spectroscopy.CD Spectra and Electrochemical Titration of CpFdFor reference, the visible CD spectra recorded during the redox titration of native CpFd are shown in Fig. 2. These data qualitatively confirm and extend previous reports (11Stephens P.J. Thomson A.J. Dunn J.B.R. Keiderling T.A. Rawlings J. Rao K.K. Hall D.O. Biochemistry. 1978; 17: 4770-4778Crossref PubMed Scopus (98) Google Scholar, 20George S.J. Thomson A.J. Crabtree D.E. Meyer J. Moulis J.-M. New J. Chem. 1991; 15: 455-465Google Scholar). The spectra of the fully oxidized protein with [4Fe-4S]2+clusters contain several moderately intense features all over the spectral range shown. Upon reduction, a significant decrease in the intensity of all these bands occurs; at −600 mV a positive band with a maximum at 360 nm and a shoulder at 380 nm is the main feature of the spectrum (Fig. 2). In contrast, the far UV region (Fig. 3) shows little, if any, change in the relatively weak negative band around 200 nm. These observations are consistent with the lack of extensive secondary structure elements in clostridial ferredoxins (21Duée E.D. Fanchon E. Vicat J. Sieker L.C. Meyer J. Moulis J.-M. J. Mol. Biol. 1994; 243: 683-695Crossref PubMed Scopus (68) Google Scholar). Moreover, they suggest that only small global structural changes are induced by adding one electron to each cluster.Figure 2CD-monitored electrochemical redox titration of CpFd (left) and CvFd (right). Spectra were recorded in an optically transparent thin layer electrode cell with a path length of 0.1 mm. Selected spectra at +90; −380 and −510 mV for CpFd and +100; −510; −610; −670 and −720 mV for CvFd (decreasing order of intensity at 412 nm, respectively) are shown. Theinsets contain Nernst curves calculated from the CD intensity at 412 nm and fitted with the following parameters: for CpFd,n = 1, E0 = −369 mV; for CvFd,n = 1, E01 = −492 mV, E02 = −663 mV.View Large Image Figure ViewerDownload (PPT)Figure 3CD spectra of CpFd (top) and CvFd (bottom) in the UV range. Solid lines, fully oxidized proteins (E = +0.1V for CpFd and −0.4 V for CvFd); broken lines, fully reduced proteins (E = −0.6 V for CpFd and −0.9 V for CvFd). The protein concentrations were 0.2 mm for CpFd and 0.15 mm for CvFd. The optical path length was 0.02 mm.View Large Image Figure ViewerDownload (PPT)The variation of the intensities of these peaks as a function of the applied potential provides a measure of the reduction potential (Fig. 2, inset). In agreement with previous studies (22Ke B. Bulen W.A. Shaw E.R. Breeze R.H. Arch. Biochem. Biophys. 1974; 162: 301-309Crossref PubMed Scopus (46) Google Scholar, 23Stombaugh N.A. Sundquist J.E. Burris R.H. Orme-Johnson W.H. Biochemistry. 1976; 15: 2633-2641Crossref PubMed Scopus (110) Google Scholar), a single redox wave, with a midpoint potential of −369 ± 4 mV, is observed between −300 and −600 mV. Moreover, the disappearance of all spectral features associated with oxidized clusters indicates that the two clusters of this protein display the same potential, as expected from a number of previous experiments (24Sweeney W.V. Rabinowitz J.C. Annu. Rev. Biochem. 1980; 49: 139-161Crossref PubMed Scopus (126) Google Scholar).CD Spectra and Electrochemical Titration of CvFdThe CD spectra of oxidized CvFd are almost identical to those of CpFd in the 300–800 nm range (Fig. 2). Therefore, the optical properties of the oxidized clusters of CvFd do not differ from typical [4Fe-4S]2+ clusters, such as those of CpFd (Fig. 2). Below 300 nm, however, significant changes between CpFd and CvFd are noticed (Fig. 3). The relatively intense negative band around 220 nm in CvFd is indicative of the presence of the α-helix evidenced in the x-ray structure of the protein (10Moulis J.-M. Sieker L.C. Wilson K.S. Dauter Z. Protein Sci. 1996; 5: 1765-1775Crossref PubMed Scopus (63) Google Scholar). This secondary structure element is absent in CpFd and has been found to produce different signatures when either the 2-turn loop or the α-helix are removed or modified in CvFd (not shown). Another difference between the two proteins occurs at 258 nm where a positive band is found in the spectra of CvFd but not in those of CpFd (Fig. 2).The reductive titration of CvFd proceeds as for CpFd with a general decrease of the CD intensity when the potential is lowered (Fig. 2). Between approximately −550 mV and −600 mV, no further significant decreases are observed (Fig. 2); a midpoint potential of −492 ± 8 mV can be calculated which agrees with values determined by electrochemical measurements (Ref. 25Smith E.T. Feinberg B.A. J. Biol. Chem. 1990; 265: 14371-14376Abstract Full Text PDF PubMed Google Scholar and see below) and redox titrations with dithionite (22Ke B. Bulen W.A. Shaw E.R. Breeze R.H. Arch. Biochem. Biophys. 1974; 162: 301-309Crossref PubMed Scopus (46) Google Scholar, 23Stombaugh N.A. Sundquist J.E. Burris R.H. Orme-Johnson W.H. Biochemistry. 1976; 15: 2633-2641Crossref PubMed Scopus (110) Google Scholar). However, the CD spectra obtained at potentials lower than this transition retain part of the features of the oxidized spectra, in contrast to what was observed with CpFd (Fig. 2). The CD spectra of CvFd did not show any decrease in intensity even after maintaining potentials in the −500 to −600 mV range for more than 1 h. Only a further decrease in potential succeeded in bleaching the CD spectra in the visible range, giving a spectroscopic signature very much like that of reduced CpFd (Fig. 2). A second midpoint redox potential at −663 ± 20 mV could be calculated from the latter part of the titration curve (Fig. 2,inset).The spectral changes associated with the first and the second transitions differ significantly (Fig. 4). The prominent CD bands at 366 (negative) and 410 nm (positive) readily disappear during the first transition. The spectral features corresponding to the second transition at lower potential are less intense and compare with the intensity observed for ferredoxins having a single [4Fe-4S] cluster (11Stephens P.J. Thomson A.J. Dunn J.B.R. Keiderling T.A. Rawlings J. Rao K.K. Hall D.O. Biochemistry. 1978; 17: 4770-4778Crossref PubMed Scopus (98) Google Scholar). A likely interpretation is that the interaction between the two [4Fe-4S] clusters, the closest metal atoms of which are less than 9 Å apart (2Dauter Z. Wilson K.S. Sieker L.C. Meyer J. Moulis J.-M. Biochemistry. 1997; 36: 16065-16073Crossref PubMed Scopus (127) Google Scholar), contributes to the CD spectra of 2[4Fe-4S] ferredoxins; the pair of CD bands at 366 and 410 nm may therefore arise from exciton coupling between the strongest transitions observed in absorption spectra around 388 nm (Fig. 5) and assigned to S-Cys ⇒ Fe charge transfer bands (26Moulis J.-M. Meyer J. Lutz M. Biochemistry. 1984; 23: 6605-6613Crossref Scopus (32) Google Scholar). It should also be noted that the orientation (Fe-S-C-C and χ1 dihedrals) of one cysteine ligand (Cys-40) is different for oxidized Cp and CvFd (10Moulis J.-M. Sieker L.C. Wilson K.S. Dauter Z. Protein Sci. 1996; 5: 1765-1775Crossref PubMed Scopus (63) Google Scholar); this may change the CD features of cluster II, coordinated by Cys-40, and may explain the spectral differences between CpFd and CvFd,i.e. the intensity of the band at 258 nm in the oxidized state and the relative intensities of the bands at 360 and 380 nm in the reduced states.Figure 4Difference CD spectra for the two redox transitions of CvFd. Solid line, spectrum at −570 mV subtracted from that recorded at −100 mV; broken line, spectrum at −720 mV subtracted from that recorded at −570 mV.View Large Image Figure ViewerDownload (PPT)Figure 5Absorption spectra of CvFd. Spectra were recorded at −200 mV, bold line; −600 mV, thin line; −730 mV, dotted line. The protein concentration was 0.8 mm; the optical path length was 0.1 mm.View Large Image Figure ViewerDownload (PPT)As in the case of CpFd the CD spectra of CvFd in the UV region remain qualitatively the same at different reduction levels (Fig. 3). This observation provides evidence that in both ferredoxins the structural changes associated with the redox interconversion of the [4Fe-4S]2+/+ clusters are minimal.At the end of the reductive titrations, increasing the potential to 0 mV restored the initial spectrum recorded for each fully oxidized protein, hence indicating that the observed changes are fully reversible and that the implemented experimental conditions are not harmful to the integrity of these 2[4Fe-4S] ferredoxins.Visible Absorption SpectroscopyIn order to sustain the above observations, the visible absorbance of CvFd was also monitored as a function of the applied potential. The decrease of absorbance at 425 nm is a measure of the degree of reduction of [4Fe-4S]2+/+ clusters, as the spectra of the reduced clusters are less intense than those of the oxidized ones (24Sweeney W.V. Rabinowitz J.C. Annu. Rev. Biochem. 1980; 49: 139-161Crossref PubMed Scopus (126) Google Scholar). Fig. 5 shows the visible absorption spectra of oxidized (applied potential, E = −200 mV), partially (E= −600 mV), and fully reduced (E = −730 mV) CvFd. Dithionite appears as effective as electrochemical reduction at −600 mV (not shown) but does not provide the largest decrease in absorbance as judged by comparison with the spectrum recorded at −730 mV. Only the latter data agree with absorbance changes observed in a sample reduced by chloroplasts under illumination (8Bachofen R. Arnon D.I. Biochim. Biophys. Acta. 1966; 120: 259-265Crossref PubMed Scopus (38) Google Scholar).Thus, the above titrations carried out with CvFd strongly suggest that the protein displays two redox transitions. Qualitatively similar results were also obtained with molecular variants of CvFd, and all reduction potential values are listed in TableII. However, such results may be biased, for instance by the involvement of a kinetic barrier slowing down the reduction of one cluster and apparently shifting the midpoint potential of the redox transition. This possibility has been addressed by additional experiments.Table IIReduction potential of CvFd and molecular variants by CD titration and electrochemical studiesFerredoxinCD titrationCyclic voltammetrySquare wave voltammetryE 1E 2E 1E 2E 1E 2CpFd−369 ± 4aWithout salt.−392b0.15 m NaCl.−405b0.15 m NaCl.CvFd−492 ± 8aWithout salt.−663 ± 20aWithout salt.−461b0.15 m NaCl.−653b0.15 m NaCl.−453b0.15 m NaCl.−658b0.15 m NaCl.Δ1c0.2 m NaCl.≈−417dBroad anodic peak; the midpoint potential was estimated from the cathodic peak position.−640—eTwo overlapping peaks observed in this potential region.−663K74−c0.2 m NaCl.−431−654−439−654Δ1K74−c0.2 m NaCl.−402≈−651dBroad anodic peak; the midpoint potential was estimated from the cathodic peak position.−424—eTwo overlapping peaks observed in this potential region.Y44Cf0.4 m NaCl.−478 ± 6−627 ± 13aWithout salt.−471≈−653dBroad anodic peak; the midpoint potential was estimated from the cathodic peak position.−468—eTwo overlapping peaks observed in this potential region.D12G−482 ± 7aWithout salt.−586 ± 11−465b0.15 m NaCl.−621b0.15 m NaCl.−453b0.15 m NaCl.−619b0.15 m NaCl.V13Gf0.4 m NaCl.−478−601−475−600Y30Fb0.15 m NaCl.−463−659−458−663a Without salt.b 0.15 m NaCl.c 0.2 m NaCl.d Broad anodic peak; the midpoint potential was estimated from the cathodic peak position.e Two overlapping peaks observed in this potential region.f 0.4 m NaCl. Open table in a new tab Reduction Potentials by Cyclic and Square Wave VoltammetriesTypical voltammograms of native CpFd and CvFd are shown in Fig. 6. CpFd shows only a single transition at approximately −400 mV, whereas CvFd shows two transitions at approximately −460 and −655 mV. The strong intensity of the single wave of CpFd agrees with the above observation that both [4Fe-4S] clusters react at this potential. Control experiments, with mediator mixtures (methyl viologen and 4′,4“-dimethyl-1′,1”trimethylene-2′,2“-dipyridinium dibromide) in the absence of protein, were carried out and showed that there is only a minor contribution of the mediator mixture to the electrochemical signal in the presence of the protein (Fig. 6). The reduction potentials determined for all forms studied are listed in Table II. From this series of measurements, it appears that the more negative value of CvFd redox potential (−655 mV) is shifted in variants with substitutions in the vicinity of cluster I, i.e. D12G and V13G; in these molecules the potential with the less negative value (−460 mV) agrees with the value measured for the native protein. Conversely, shifts of the less negative but not of the more negative redox potential were observed in variants, such as Δ1, K74−, and Δ1K74−, structurally modified around cluster II. These data indicate that the redox transition at −460 mV (native CvFd) arises from cluster II and the lowest transition at −655 mV is due to cluster I. Proteins carrying the C-terminal truncation displayed partially broadened and split electrochemical signals, so that some potentials could not be determined reliably. This electrochemical behavior correlates with a decreased stability of these proteins which was also observed during the CD-monitored redox titrations and other experiments (not shown). No significant changes were found for Y30F, as already observed with similar variants of other ferredoxins (27Quinkal I. Kyritsis P. Kohzuma T. Im S.-C. Sykes A.G. Moulis J.-M. Biochim. Biophys. Acta. 1996; 1295: 201-208Crossref PubMed Scopus (14) Google Scholar).Figure 6Electrochemical characterization of CpFd and CvFd. Cyclic (left) and square wave (right) voltammograms of the buffer control with viologen mediators added (top), CpFd (middle), and CvFd (bottom). Cyclic voltammograms were taken at 25 °C and at a potential scan rate of 10 mV/s. Square wave voltammograms were taken at 25 °C and a frequency of 8 Hz with a step potential of 5 mV and an amplitude of 50 mV.View Large Image Figure ViewerDownload (PPT)Kinetics of the Interaction between the Electrode and the Protein2[4Fe-4S] ferredoxins show quasi-reversible electrode kinetics at the gold electrode in the presence of viologens up to scan rates of 1 V/s. At high scan rates, the separation between the anodic and the cathodic peak increased with increasing scan rate; from this, the heterogenous electron transfer rate constant k0(28Nicholson R.S. Anal. Chem. 1965; 37: 1351-1355Crossref Scopus (2983) Google Scholar) could be estimated (Table III), assuming a diffusion coefficient D = 10−6cm2/s which is consistent with the observed peak current. Quite remarkably, different electrode kinetics were observed for cluster I (E m = −655 mV) and cluster II (E m = −460 mV); the heterogenous rate constant for the oxido-reduction of the latter was determined as approximately 4 × 10−3 cm/s, whereas the former showed reversible electrode kinetics up to scan rates of 0.5 V/s. Therefore, a lower limit of 2 × 10−2 cm/s was estimated for the heterogenous rate constant of cluster I in all variants studied. These values support the statement that no fast electron transfer takes place between the clusters (7Huber J.G. Gaillard J. Moulis J.-M. Biochemistry. 1995; 34: 194-205Crossref PubMed Scopus (45) Google Scholar); otherwise, oxidoreduction of both clusters could proceed rapidly via cluster I.Table IIIKinetics of reduction of native CvFd and molecular variantsFerredoxinHeterogenous electron transfer rate, k0 of cluster IIHomogeneous rate constant for the reduction (of cluster II) by dithionitecm/sm−1/2 s−1Native CvFd4 · 10−30.02Y30F(4–8) · 10−30.02Y44S0.03Y44C11 · 10−3D12G8 · 10−30.03K74E ≫0.2K74−3 · 10−3 ≫0.2Δ1>2 · 10−2 ≫0.2Δ1K74−>10−2 ≫0.2CpFd6 · 10−319aValue from data obtained by stopped-flow measurements (52).a Value from data obtained by stopped-flow measurements (52Lambeth D.O. Palmer G. J. Biol. Chem. 1973; 248: 6095-6103Abstract Full Text PDF PubMed Google Scholar). Open table in a new tab Electron transfer to cluster II is faster in the Δ1 and Δ1K74− forms in which it is comparable to electron transfer to cluster I. This result is consistent with dithionite reduction kinetics discussed below. The other structural changes studied in this work had less significant effects on the electron transfer kinetics except for Y44C which displayed an increased heterogenous rate constant by a factor of approximately 3-fold. It is conceivable that cysteine in this position facilitates electron transfer to and from cluster II by interacting with the gold electrode or that the removal of the aromatic ring makes cluster II more exposed.Surprisingly, the heterogenous rate constant determined for CpFd, which agrees with previously reported ones (29Van Dijk C. van Eijs T. van Leeuwen J.W. Veeger C. FEBS Lett. 1984; 166: 76-80Crossref Scopus (26) Google Scholar, 30Armstrong F.A. Cox P.A. Hill H.A.O. Lowe V.J. Oliver B.N. J. Electroanal. Chem. 1987; 217: 331-366Crossref Scopus (167) Google Scholar), is closer to those of CvFd cluster II than to those of CvFd cluster I (Table III). This is another noteworthy difference between the latter and CpFd clusters, despite their structural similarity (10Moulis J.-M. Sieker L.C. Wilson K.S. Dauter Z. Protein Sci. 1996; 5: 1765-1775Crossref PubMed Scopus (63) Google Scholar).Effect of Ionic Strength and pHThe effect of ionic strength on the values of the reduction potential of native CvFd has been measured in solutions with NaCl concentrations up to 1 m. A linear relationship between the redox potential and the square root of the ionic strength was observed over the whole range studied with a slope of +35 mV/m0.5 for both clusters. The variation is similar to that obtained in previous studies on [4Fe-4S]2+/+ clusters in other proteins (23Stombaugh N.A. Sundquist J.E. Burris R.H. Orme-Johnson W.H. Biochemistry. 1976; 15: 2633-2641Crossref PubMed Scopus (110) Google Scholar).The effect of pH has also been studied. At pH values equal to or below 6, the electrochemical response was not satisfactory in the implemented conditions. Although the ionic strength was not kept constant at the different pH values, the weak dependence with ionic strength observed at pH 7.5 dismisses a major contribution of this parameter. Only at pH values above 10 do the reduction potentials of both transitions significantly shift; these data may indicate the binding of hydroxide ions to the clusters and associated changes in their electronic properties. At these high pH values, structural modifications of the protein must also be considered. The case of cluster II is interesting because, in contrast to cluster I, this group displays an increase of the redox potential with increasing pH opposite to the effect of electrostatic repulsion between the [Fe4S4(Cys)4]2−/3−cluster and nearby deprotonated residues, such as His-43.Kinetics of Dithionite Reduction of CvFdFrom the comparison of the absorption spectra described above, dithionite appears to be able to reduce only cluster II in CvFd. The decrease with time of the absorbance at 425 nm has been analyzed as a pseudo first-order kinetic process; the rate constants calculated using the added dithionite concentration are listed in Table III. It is easily concluded that in the molecular variants missing either loop Δ1 or half of the C terminus α-helix, reduction of cluster" @default.
• W2091880825 created "2016-06-24" @default.
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• W2091880825 date "1998-06-01" @default.
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• W2091880825 title "The Two [4Fe-4S] Clusters in Chromatium vinosumFerredoxin Have Largely Different Reduction Potentials" @default.
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