Comparison of redox and EPR properties of the molybdenum iron proteins of Clostridium pasteurianum and Azotobacter vinelandii nitrogenases

Both heterologous crosses of the Clostridium pasteurianum and Azotobacter vinelandii nitrogenase components are completely inactive, although the reasons for this incompatibility are not known. We have compared a number of properties of the MoFe proteins from these organisms (Cp1 and Av1, respectively) in an attempt to find differences that could explain this lack of functional activity. Optical and CD spectroscopic titrations are similar for both Av1 and Cp1, but EPR titrations are significantly different, suggesting different chemical reactivity patterns and/or magnetic interaction behavior. Similarly, reduction measurements on the six-electron-oxidized state of Cp1 and Av1 at controlled potentials indicate a difference in both the relative reduction sequence of the redox centers and the numerical values for their measured midpoint potentials. EPR measurements as a function of temperature also demonstrate that the relaxation behavior of the S = 3/2 MoFe centers associated with the proteins differ markedly. The Cp1 EPR signal only begins to undergo broadening above 65 K, whereas the Av1 signal is severely broadened above 25 K. These variations in the EPR properties for the two proteins are not likely to be due to differences in the stoichiometry and/or geometry of the MoFe cluster units themselves since similar EPR studies of the isolated cofactors showed them to be essentially identical. Thus, the different EPR behavior of the two proteins seems to arise either from protein constraints imposed on identical cofactors, and/or from magnetic interactions due to neighboring metal clusters.     

The electronic state of heme in cytochrome oxidase II. Oxidation-reduction potential interactions and heme iron spin state behavior observed in reductive titrations.

Magnetic circular dichroism (MCD), electron paramagnetic resonance (EPR), and optical absorption spectroscopies have been used to monitor the concentrations of oxidized and reduced heme and copper during stoichiometric reductive titrations of purified beef heart cytochrome oxidase. The MCD data are deconvoluted to obtain the concentrations of reduced cytochromes a and a3 during the titrations; analysis of the EPR spectra provides complementary data on the concentrations of the EPR-detectable species. For the native enzyme in the absence of exogenous ligands, cytochromes a and a3 are reduced to approximately the same extent at all points in the titration. The reduction of the EPR-detectable copper, on the other hand, initially lags the reduction of the two cytochromes but in the final stages of the titration is completely reduced prior to either cytochrome a or a3. These non-Nernstian titration results are interpreted to indicate that the primary mode of heme-heme interaction in cytochrome oxidase involves shifts in oxidation-reduction potential for each of the two cytochromes such that a change in oxidation state for one of the hemes lowers the oxidation-reduction potential of the second heme by approximately 135 mV. In these titrations high spin species are detected which account for 0.25 spin/oxidase maximally. Evidence is presented to indicate that at least some of these signals can be attributed to cytochrome a3+ which has undergone a low-spin to high-spin state transition in the course of the titration. In the presence of carbon monoxide the oxidation-reduction properties of cytochromes a and a3 are markedly altered. The a32+. CO complex is fully formed prior to reduction of either cytochrome a3+ or the EPR-detectable copper. The g = 3 EPR signal attributed to cytochrome a3+ decreases as the MCD intensity of cytochrome a2+ increases; no significant high-spin intensity is observed at any intermediate stage of reduction. We interpret these Nernstian titration results to indicate that in the presence of ligands the oxidation-reduction potential of cytochrome a relative to cytochrome a3 is determined by the oxidation-reduction state of the stabilized cytochrome a3 ligand complex; if ligand binding occurs to reduced cytochrome a3 then cytochrome a titrates with a lower potential; cytochrome a titrates with a higher potential if oxidized cytochrome a3 is stabilized by ligand binding.     

Local anesthetic-induced microscopic and mesoscopic effects in micelles. A fluorescence, spin label and SAXS study. Single angle X-ray scattering

The interaction of the local anesthetic tetracaine (TTC) with anionic sodium lauryl sulfate (SLS) and zwitterionic 3-(N-hexadecyl-N,N-dimethylammonio)propanesulfonate (HPS) micelles was investigated by fluorescence, spin labeling EPR and small angle X-ray scattering (SAXS). Fluorescence pH titrations allowed the choice of adequate pHs for the EPR and SAXS experiments, where either charged or uncharged TTC would be present. The data also indicated that the anesthetic is located in a less polar environment than its charged counterpart in both micellar systems. EPR spectra evidenced that both anesthetic forms increased molecular organization within the SLS micelle, the cationic form exerting a more pronounced effect. The SAXS data showed that protonated TTC causes an increase in the SLS polar shell thickness, hydration number, and aggregation number, whereas the micellar features are not altered upon incorporation of the uncharged drug. The combined results suggest that the electrostatic interaction between charged TTC and SLS, and the intercalation of the drug in the micellar polar region induce a change in molecular packing with a decrease in the mean cross-sectional area, not observed when the neutral drug sinks more deeply into the micellar hydrophobic domain. In the case of HPS micelles, the EPR spectral changes were small for the charged anesthetic and the SAXS data did not evidence any change in micellar structure, suggesting that this species protrudes more into the aqueous phase due to the lack of electrostatic attractive forces in this system.     

Defining the Q-site of Escherichia coli fumarate reductase by site-directed mutagenesis, fluorescence quench titrations and EPR spectroscopy

We have used fluorescence quench titrations, EPR spectroscopy and steady-state kinetics to study the effects of site-directed mutants of FrdB, FrdC and FrdD on the proximal menaquinol (MQH(2)) binding site (Q(P)) of Escherichia coli fumarate reductase (FrdABCD) in cytoplasmic membrane preparations. Fluorescence quench (FQ) titrations with the fluorophore and MQH(2) analog 2-n-heptyl-4-hydroxyquinoline-N-oxide (HOQNO) indicate that the Q(P) site is defined by residues from FrdB, FrdC and FrdD. In FQ titrations, wild-type FrdABCD binds HOQNO with an apparent K(d) of 2.5 nM, and the following mutations significantly increase this value: FrdB-T205H (K(d) = 39 nM); FrdB-V207C (K(d) = 20 nM); FrdC-E29L (K(d) = 25 nM); FrdC-W86R (no detectable binding); and FrdD-H80K (K(d) = 20 nM). In all titrations performed, data were fitted to a monophasic binding equation, indicating that no additional high-affinity HOQNO binding sites exist in FrdABCD. In all cases where HOQNO binding is detectable by FQ titration, it can also be observed by EPR spectroscopy. Steady-state kinetic studies of fumarate-dependent quinol oxidation indicate that there is a correlation between effects on HOQNO binding and effects on the observed K(m) and k(cat) values, except in the FrdC-E29L mutant, in which HOQNO binding is observed, but no enzyme turnover is detected. In this case, EPR studies indicate that the lack of activity arises because the enzyme can only remove one electron from reduced MQH(2), resulting in it being trapped in a form with a bound menasemiquinone radical anion. Overall, the data support a model for FrdABCD in which there is a single redox-active and dissociable Q-site.     

Local anesthetic-induced microscopic and mesoscopic effects in micelles. A fluorescence,spin label and SAXS study

The interaction of the local anesthetic tetracaine (TTC) with anionic sodium lauryl sulfate (SLS) and zwitterionic 3-(N-hexadecyl-N,N-dimethylammonio)propanesulfonate (HPS) micelles was investigated by fluorescence, spin labeling EPR and small angle X-ray scattering (SAXS). Fluorescence pH titrations allowed the choice of adequate pHs for the EPR and SAXS experiments, where either charged or uncharged TTC would be present. The data also indicated that the anesthetic is located in a less polar environment than its charged counterpart in both micellar systems. EPR spectra evidenced that both anesthetic forms increased molecular organization within the SLS micelle, the cationic form exerting a more pronounced effect. The SAXS data showed that protonated TTC causes an increase in the SLS polar shell thickness, hydration number, and aggregation number, whereas the micellar features are not altered upon incorporation of the uncharged drug. The combined results suggest that the electrostatic interaction between charged TTC and SLS, and the intercalation of the drug in the micellar polar region induce a change in molecular packing with a decrease in the mean cross-sectional area, not observed when the neutral drug sinks more deeply into the micellar hydrophobic domain. In the case of HPS micelles, the EPR spectral changes were small for the charged anesthetic and the SAXS data did not evidence any change in micellar structure, suggesting that this species protrudes more into the aqueous phase due to the lack of electrostatic attractive forces in this system.     

Iron entry route in horse spleen apoferritin. Involvement of the three-fold channels as probed by selective reaction of cysteine-126 with the spin label 4-maleimido-tempo.

Apoferritin has been selectively labeled with a maleimide nitroxide derivative at Cys-126, located in the hydrophilic 3-fold channels. Titration of this derivative with Fe(II), which gives rise to the initial Fe(III)-apoferritin complex, produces, at low metal-to-protein ratios, a decrease of the intensity of the label EPR signal due to the occurrence of a magnetic dipolar interaction. A label-metal distance ranging between 8-12 A can be estimated from titrations performed with VO(IV), which is known to bind in the 3-fold channels, and likewise produces a decrease in the label EPR signal. The present findings indicate that iron binds in the hydrophilic channels in its higher oxidation state and that these channels represent the metal entry route at least at low metal-to-protein ratios.     

CO dehydrogenase from Clostridium thermoaceticum. EPR and electrochemical studies in CO2 and argon atmospheres

The EPR and redox properties of the metal complexes in CO dehydrogenase (CODH) from Clostridium thermoaceticum were studied. Controlled potential coulometric reductive titrations of CODH were performed under argon and CO2 atmospheres. In the titrations performed under argon, five to eight electrons/dimer were required for reduction, and four distinct EPR signals appeared. These included a signal with gave = 1.82 (Em approximately -220 mV), two signals with the same g values but different linewidths at gave = 1.94 (Em approximately -440 mV), and a signal at gave = 1.86 (Em approximately -530 mV). All of the S = 1/2 EPR signals had low spin concentrations; values between 0.2 and 0.3 spins/dimer were typically obtained for each signal. Features between g = 6 and 4, typical of S = 3/2 states, were also observed, and these may account, at least to some degree, for the low spin concentration values. Under CO2, and at negative potentials, CODH served as an electrocatalyst in the reduction of CO2 to CO. The apparent half-maximal activity for this reduction at pH 6.3 occurred at -430 mV, a potential near the thermodynamic value. An EPR signal, arising from a complex containing Ni, Fe, and the carbon from CO/CO2 developed along with this activity. The reduction of this complex is probably the last step to occur prior to the catalysis of CO2 reduction.     

The cytochrome bc1 complex from Rhodovulum sulfidophilum is a dimer with six quinones per monomer and an additional 6-kDa component.

A highly active, large-scale preparation of cytochrome bc1 complex has been obtained from the photosynthetic purple bacterium Rhodovulum (Rhv.) sulfidophilum. It has been characterized using mass spectrometry, quinone and lipid analysis as well as inhibitor binding. About 35 mg of pure complex can be obtained from 1 g of membrane protein. EPR spectroscopy and optical titrations have been used to obtain the redox midpoint potentials of the cofactors. The Em-value of 310 mV for the Rieske protein is the most positive midpoint potential for this protein in a bc1 complex so far. The bc1 complex from Rhv. sulfidophilum is very stable and consists of three subunits and a 6-kDa polypeptide. The complex appears as a dimer in solution and contains six quinone molecules per monomer which are tightly bound. EPR spectroscopy shows that the Q(o) site is highly occupied. High detergent concentrations convert the complex into an inactive, monomeric form that has lost the Rieske protein as well as the quinones and the 6-kDa component.     

The redox properties of the iron-sulphur cluster in hydrogenase from Chromatium vinosum, strain D

The midpoint potentials of the changes in the electron spin resonance (ESR) spectra in the region of g = 2 in hydrogenase II from Chromatium vinosum were estimated by redox titrations. As the enzyme was progressively reduced, the g = 2.02 signal increased, while the satellite lines at g = 1.98 etc. decreased. At still lower potentials the signal at g = 2.02 decreased. The midpoint potentials of the two processes were estimated to be + 100 mV and - 20 mV, respectively, at pH 8.5. The first potential showed significant pH-dependence. The titration data fitted to n = 1 curves with reasonable reversibility. The enzyme activity showed no significant changes in this potential range. The results are discussed in relation to the interaction of the iron-sulphur cluster with nickel.     

Impact of mutations on the midpoint potential of the [4Fe-4S]+1,+2 cluster and on catalytic activity in electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO)

Electron-transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO) is an iron-sulfur flavoprotein that accepts electrons from electron-transfer flavoprotein (ETF) and reduces ubiquinone from the Q-pool. ETF-QO contains a single [4Fe-4S]2+,1+ cluster and one equivalent of FAD, which are diamagnetic in the isolated oxidized enzyme and can be reduced to paramagnetic forms by enzymatic donors or dithionite. Mutations were introduced by site-directed mutagenesis of amino acids in the vicinity of the iron-sulfur cluster of Rhodobacter sphaeroides ETF-QO. Y501 and T525 are equivalent to Y533 and T558 in the porcine ETF-QO. In the porcine protein, these residues are within hydrogen-bonding distance of the Sgamma of the cysteine ligands to the iron-sulfur cluster. Y501F, T525A, and Y501F/T525A substitutions were made to determine the effects on midpoint potential, activity, and EPR spectral properties of the cluster. The integrity of the mutated proteins was confirmed by optical spectra, EPR g-values, and spin-lattice relaxation rates, and the cluster to flavin point-dipole distance was determined by relaxation enhancement. Potentiometric titrations were monitored by changes in the CW EPR signals of the cluster and semiquinone. Single mutations decreased the midpoint potentials of the iron-sulfur cluster from +37 mV for wild type to -60 mV for Y501F and T525A and to -128 mV for Y501F/T525A. Lowering the midpoint potential resulted in a decrease in steady-state ubiquinone reductase activity and in ETF semiquinone disproportionation. The decrease in activity demonstrates that reduction of the iron-sulfur cluster is required for activity. There was no detectable effect of the mutations on the flavin midpoint potentials.     

EPR determination of interaction redox potentials in a multiheme cytochrome: cytochrome c3 from Desulfovibrio desulfuricans Norway

In cytochromes c3 which contain four hemes per molecule, the redox properties of each heme may depend upon the redox state of the others. This effect can be described in terms of interaction redox potentials between the hemes and must be taken into account in the characterization of the redox properties of the molecule. We present here a method of measurement of these interactions based on the EPR study of the redox equilibria of the protein. The microscopic and macroscopic midpoint potentials and the interaction potentials are deduced from the analysis of the redox titration curves of the intensity and the amplitude of the EPR spectrum. This analysis includes a precise simulation of the spectrum of the protein in the oxidized state in order to determine the relative contribution of each heme to the spectral amplitude. Using our method on cytochrome c3 from D. desulfuricans Norway, we found evidence for the existence of weak interaction potentials between the hemes. The three interaction potentials which have been measured are characterized by absolute values lower than 20 mV in contrast with the values larger than 40-50 mV which have been reported for cytochrome c3 from D. gigas. Simulations of the spectra of samples poised at different potentials indicate a structural modification of the heme with the most negative potential during the first step of reduction. The correspondence between the redox sites as characterized by the EPR potentiometric titration and the hemes in the tridimensional structure is discussed.     

Stoichiometry and spectral properties of the MoFe cofactor and noncofactor redox centers in the MoFe protein of nitrogenase from Azotobacter vinelandii

Reductive EPR and optical titrations of oxidized MoFe protein using reduced methyl viologen as reductant were used to quantitate the stoichiometry of the various spectroscopically and electrochemically distinct redox centers in the oxidized MoFe protein. Three centers were found to correlate with the EPR signal development (MoFe cofactor centers), and three centers were found to be independent of the EPR signal (P clusters) but to demonstrate distinct optical and kinetic properties. Oxidative EPR and optical titrations of reduced MoFe protein are reported which support the presence of three P-cluster centers. The optical titrations show a distinct change in kinetic behavior between the MoFe cofactor and P-cluster centers. Controlled potential coulometry demonstrates that incremental oxidation of reduced protein by methylene blue, thionine, or indigodisulfonate occurs specifically at three P-cluster sites. Subsequent oxidation by methylene blue and thionine (but not indigodisulfonate) causes the EPR signal to disappear. Three P-cluster sites, two EPR sites, and one presently uncharacterized site are suggested by the results of this study.     

Characterization of arsenite-complexed xanthine oxidase at room temperature. Spectral properties and pH-dependent redox behavior of the molybdenum-arsenite center

Several aspects of the interaction of xanthine oxidase with arsenite are investigated. Room temperature potentiometric titrations using EPR to monitor Molybdenum reduction reveal midpoint potentials of -225 mV for the Mo(VI)-arsenite/Mo(V)-arsenite couple and -440 mV for the Mo(V)-arsenite/Mo(IV)-arsenite couple at pH 8.3. Under the same conditions, the values for native enzyme are -395 mV and -420 mV, respectively. The predicted effects of the altered Mo(VI)/Mo(V) potential on the distributions of reducing equivalents in partially reduced enzyme are compared with the experimentally observed effects in optical experiments. The bleaching that occurs on reduction of the chromophore that is generated when arsenite binds to oxidized enzyme is characterized and found to be associated with reduction of Mo(V)-arsenite to Mo(V)-arsenite. This probe enables determination of the midpoint potential for this conversion using optical data. From such data at a series of pH values ranging from 6.15 to 9.9, a pH dependence of -60 mV/pH unit increase is determined for this couple above pH 7. The ability of arsenite to bind to reduced xanthine oxidase and to desulfo enzyme are also investigated. Reduced active enzyme binds arsenite much more tightly (Kd less than 0.1 microM) and more rapidly than does oxidized active enzyme (Kd = 8 microM); oxidized desulfo enzyme binds arsenite almost as tightly (Kd = 20 microM) as does the oxidized active enzyme.     

The dinuclear iron-oxo ferroxidase center of Pyrococcus furiosus ferritin is a stable prosthetic group with unexpectedly high reduction potentials

Recombinant ferritin from Pyrococcus furiosus expressed in Escherichia coli exhibits in EPR monitored redox titrations a mixed valence (Fe(3+)-Fe2+) S=1/2 signal at intermediate potentials that is a high-resolution homolog of the ferroxidase signal previously described for reconstituted horse spleen apo-ferritin. P. furiosus reconstituted apo-ferritin reduced to intermediate potentials exhibits the same mixed-valence signal, which integrates to close to one spin per subunit. The reduction potentials of +210 and +50 mV imply that the iron dimer is a stable prosthetic group with three redox states.     

Characterization of the potentiometric behavior of soluble cytochrome oxidase by magnetic circular dichroism. Evidence in support of heme-heme interaction

Magnetic circular dichroism spectroscopy has been used to characterize the oxidation-reduction behavior of cytochromes a and a3 during potentiometric experiments. The experiments were complicated by the presence of slow, time-dependent changes during reduction, and it appears that reliable results can only be obtained during reoxidation or with enzyme that has been subjected to a cycle of reduction and oxidation. Under all experimental conditions the reduction levels of cytochrome a and a3 are comparable. This result cannot be reconciled with a model in which the two heme centers have defined and well resolved potentials. The most straightforward explanation of the data requires an oxidation-reduction coupling of the potentials of the two hemes, i.e. an allosteric or heme-heme interaction, which is about 2 K cal/mol in magnitude. There is a good correlation between magnetic circular dichroism and EPR measurements obtained on parallel samples. The kinetics of the slow, time-dependent processes have been characterized by measurement of a variety of spectral properties and enzyme activity. All parameters measured change at comparable rates, implying a common rate-controlling event. A new copper EPR signal has been observed at high pH. This signal appears to rise from the "EPR-undetectable" copper center.     

Potentiometric studies on yeast complex III

Potentiometric measurements have been performed on Complex III from bakers' yeast. The midpoint potentials for the b and c cytochromes were measured using room-temperature MCD and liquid-helium temperature EPR. A value of 270 mV was obtained for cytochrome c1, regardless of temperature, while the midpoint potentials found for the two species of cytochrome b varied with temperatures, viz., 62 and -20 mV at room temperature (MCD) compared to 116 and -4 mV at about 10 K (EPR). The midpoint potential of the iron-sulfur center obtained by low-temperature EPR was 286 mV. An abrupt conformational change occurred immediately after this center was fully reduced resulting in a change in EPR line shape. The potentials of the two half-reactions of ubiquinone were measured by following the semiquinone radical signal at 110 K and 23 degrees C. Potentials of 176 and 51 mV were found at low temperature, while values of 200 and 110 mV were observed at room temperature. The midpoint potential of cytochrome c1 was found to be pH independent. The potentials of cytochrome b were also independent of pH when titrations were performed in deoxycholate buffers, while a variation of -30 mV per pH unit was observed for both cytochrome c species in taurocholate buffers. These two detergents also produced different MCD contributions of the two b cytochromes. A decrease in Em of greater than 300 mV was found in potentiometric measurements of cytochrome c1 at high ratios of dye to Complex III. Antimycin does not affect the redox potentials of cytochrome c1 but appears to induce a transition of the low-potential b heme to a high-potential species. This transition is mediated by ubiquinone.     

EPR and redox characterization of iron-sulfur centers in nitrate reductases A and Z from Escherichia coli. Evidence for a high-potential and a low-potential class and their relevance in the electron-transfer mechanism.

The redox properties of the iron-sulfur centers of the two nitrate reductases from Escherichia coli have been investigated by EPR spectroscopy. A detailed study of nitrate reductase A performed in the range +200 mV to -500 mV shows that the four iron-sulfur centers of the enzyme belong to two classes with markedly different redox potentials. The high-potential group comprises a [3Fe-4S] and a [4Fe-4S] cluster whose midpoint potentials are +60 mV and +80 mV, respectively. Although these centers are magnetically isolated, they are coupled by a significant anticooperative redox interaction of about 50 mV. The [4Fe-4S]1+ center occurs in two different conformations as shown by its composite EPR spectrum. The low-potential group contains two [4Fe-4S] clusters with more typical redox potentials (-200 mV and -400 mV). In the fully reduced state, the three [4Fe-4S]1+ centers are magnetically coupled, leading to a broad featureless spectrum. The redox behaviour of the high-pH EPR signal given by the molybdenum cofactor was also studied. The iron-sulfur centers of the second nitrate reductase of E. coli, nitrate reductase Z, exhibit essentially the same characteristics than those of nitrate reductase A, except that the midpoint potentials of the high-potential centers appear negatively shifted by about 100 mV. From the comparison between the redox centers of nitrate reductase and of dimethylsulfoxide reductase, a correspondence between the high-potential iron-sulfur clusters of the two enzymes can be proposed.     

Spectroscopic studies of the molybdenum-containing dimethyl sulfoxide reductase from Rhodobacter sphaeroides f. sp. denitrificans.

Absorption and EPR spectroscopic properties of purified dimethyl sulfoxide (Me2SO) reductase from Rhodobacter sphaeroides f. sp. denitrificans have been examined. The absence of prosthetic groups other than the molybdenum center in the enzyme has made it possible to study its absorption properties. The enzyme displays multiple absorbance peaks in both the oxidized and the dithionite-reduced forms. The oxidized enzyme has absorbance peaks at 280, 350, 470, 550, and 720 nm while the dithionite-reduced enzyme has peaks at 280, 374, and 645 nm with a shoulder at 430 nm. A comparison of the absorbance spectrum of oxidized Me2SO reductase with that of the molybdenum fragment of rat liver sulfite oxidase shows that the 350 and 470 peaks are common to both proteins. EPR studies of the Mo(V) form of Me2SO reductase show a rhombic signal with g1 = 1.988, g2 = 1.977, g3 = 1.961, and g(ave) = 1.975. The signal shows evidence of coupling to an exchangeable proton with A1 = 1.05, A2 = 1.13, A3 = 0.98, and Aave = 1.05 millitesla. These parameters are similar to those of other Mo enzymes, however, the epr signal of this enzyme differs from those of other Mo hydroxylases in showing only a slight sensitivity to pH and no detectable anion effect. EPR potentiometric titrations of Me2SO reductase gave midpoint potentials of +144 mV for the Mo(VI)/Mo(V) couple and +160 mV for the Mo(V)/Mo(IV) couple at room temperature and +141 mV for the Mo(VI)/Mo(V) couple and +200 mV for the Mo(V)/Mo(IV) couple at 173 K.     

The membrane-bound [NiFe]-hydrogenase (Ech) from Methanosarcina barkeri: unusual properties of the iron-sulphur clusters.

The purified membrane-bound [NiFe]-hydrogenase from Methanosarcina barkeri was studied with electron paramagnetic resonance (EPR) focusing on the properties of the iron-sulphur clusters. The EPR spectra showed signals from three different [4Fe-4S] clusters. Two of the clusters could be reduced under 101 kPa of H2, whereas the third cluster was only partially reduced. Magnetic interaction of one of the clusters with an unpaired electron localized on the Ni-Fe site indicated that this was the proximal cluster as found in all [NiFe]-hydrogenases. Hence, this cluster was assigned to be located in the EchC subunit. The other two clusters could therefore be assigned to be bound to the EchF subunit, which has two conserved four-Cys motifs for the binding of a [4Fe-4S] cluster. Redox titrations at different pH values demonstrated that the proximal cluster and one of the clusters in the EchF subunit had a pH-dependent midpoint potential. The possible relevance of these properties for the function of this proton-pumping [NiFe]-hydrogenase is discussed.