The [NiFe] hydrogenase from Allochromatium vinosum studied in EPR-detectable states: H/D exchange experiments that yield new information about the structure of the active site.

In this study we report on thus-far unobserved proton hyperfine couplings in the well-known EPR signals of [NiFe] hydrogenases. The preparation of the enzyme in several highly homogeneous states allowed us to carefully re-examine the Ni(u)*, Ni(r)*, Ni(a)-C* and Ni(a)-L* EPR signals which are present in most [NiFe] hydrogenases. At high resolution (modulation amplitude 0.57 G), clear indications for hyperfine interactions were observed in the g(z) line of the Ni(r)* EPR signal. The hyperfine pattern became more pronounced in 2H2O. Simulations of the spectra suggested the interaction of the Ni-based unpaired electron with two equivalent, non-exchangeable protons (A1,2=13.2 MHz) and one exchangeable proton (A3=6.6 MHz) in the Ni(r)* state. Interaction with an exchangeable proton could not be observed in the Ni(u)* EPR signal. The identity of the three protons is discussed and correlated to available ENDOR data. It is concluded that the NiFe centre in the Ni(r)* state contains a hydroxide ligand bound to the nickel, which is pointing towards the gas channel rather than to iron.

Involvement of hyp gene products in maturation of the H(2)-sensing [NiFe] hydrogenase of Ralstonia eutropha.

The biosynthesis of [NiFe] hydrogenases is a complex process that requires the function of the Hyp proteins HypA, HypB, HypC, HypD, HypE, HypF, and HypX for assembly of the H(2)-activating [NiFe] site. In this study we examined the maturation of the regulatory hydrogenase (RH) of Ralstonia eutropha. The RH is a H(2)-sensing [NiFe] hydrogenase and is required as a constituent of a signal transduction chain for the expression of two energy-linked [NiFe] hydrogenases. Here we demonstrate that the RH regulatory activity was barely affected by mutations in hypA, hypB, hypC, and hypX and was not substantially diminished in hypD- and hypE-deficient strains. The lack of HypF, however, resulted in a 90% decrease of the RH regulatory activity. Fourier transform infrared spectroscopy and the incorporation of (63)Ni into the RH from overproducing cells revealed that the assembly of the [NiFe] active site is dependent on all Hyp functions, with the exception of HypX. We conclude that the entire Hyp apparatus (HypA, HypB, HypC, HypD, HypE, and HypF) is involved in an efficient incorporation of the [NiFe] center into the RH.

A paramagnetic species with unique EPR characteristics in the active site of heterodisulfide reductase from methanogenic archaea.

Heterodisulfide reductase (Hdr) from methanogenic archaea is an iron-sulfur protein that catalyses the reversible reduction of the heterodisulfide (CoM-S-S-CoB) of the methanogenic thiol coenzymes, coenzyme M (H-S-CoM) and coenzyme B (H-S-CoB). In EPR spectroscopic studies with the enzyme from Methanothermobacter marburgensis, we have identified a unique paramagnetic species that is formed upon reaction of the oxidized enzyme with H-S-CoM in the absence of H-S-CoB. This paramagnetic species can be reduced in a one-electron step with a midpoint-potential of -185 mV but not further oxidized. A broadening of the EPR signal in the 57Fe-enriched enzyme indicates that it is at least partially iron based. The g values (gxyz = 2.013, 1.991 and 1.938) and the midpoint potential argue against a conventional [2Fe-2S]+, [3Fe-4S]+, [4Fe-4S]+ or [4Fe-4S]3+ cluster. This species reacts with H-S-CoB to form an EPR silent form. Hence, we propose that only a half reaction is catalysed in the presence of H-S-CoM and that a reaction intermediate is trapped. This reaction intermediate is thought to be a [4Fe-4S]3+ cluster that is coordinated by one of the cysteines of a nearby active-site disulfide or by the sulfur of H-S-CoM. A paramagnetic species with similar EPR properties was also identified in Hdr from Methanosarcina barkeri.

A stable organic free radical in anaerobic benzylsuccinate synthase of Azoarcus sp. strain T.

The novel enzyme benzylsuccinate synthase initiates anaerobic toluene metabolism by catalyzing the addition of toluene to fumarate, forming benzylsuccinate. Based primarily on its sequence similarity to the glycyl radical enzymes, pyruvate formate-lyase and anaerobic ribonucleotide reductase, benzylsuccinate synthase was speculated to be a glycyl radical enzyme. In this report we use EPR spectroscopy to demonstrate for the first time that active benzylsuccinate synthase from the denitrifying bacterium Azoarcus sp. strain T harbors an oxygen-sensitive stable organic free radical. The EPR signal of the radical was centered at g = 2.0021 and was characterized by a major 2-fold splitting of about 1.5 millitesla. The strong similarities between the EPR signal of the benzylsuccinate synthase radical and that of the glycyl radicals of pyruvate formate-lyase and anaerobic ribonucleotide reductase provide evidence that the benzylsuccinate synthase radical is located on a glycine residue, presumably glycine 828 in Azoarcus sp. strain T benzylsuccinate synthase.

The H2 sensor of Ralstonia eutropha. Biochemical characteristics, spectroscopic properties, and its interaction with a histidine protein kinase.

Previous genetic studies have revealed a multicomponent signal transduction chain, consisting of an H(2) sensor, a histidine protein kinase, and a response regulator, which controls hydrogenase gene transcription in the proteobacterium Ralstonia eutropha. In this study, we isolated the H(2) sensor and demonstrated that the purified protein forms a complex with the histidine protein kinase. Biochemical and spectroscopic analysis revealed that the H(2) sensor is a cytoplasmic [NiFe]-hydrogenase with unique features. The H(2)-oxidizing activity was 2 orders of magnitude lower than that of standard hydrogenases and insensitive to oxygen, carbon monoxide, and acetylene. Interestingly, only H(2) production but no HD formation was detected in the D(2)/H(+) exchange assay. Fourier transform infrared data showed an active site similar to that of standard [NiFe]-hydrogenases. It is suggested that the protein environment accounts for a restricted gas diffusion and for the typical kinetic parameters of the H(2) sensor. EPR analysis demonstrated that the [4Fe-4S] clusters within the small subunit were not reduced under hydrogen even in the presence of dithionite. Optical spectra revealed the presence of a novel, redox-active, n = 2 chromophore that is reduced by H(2). The possible involvement of this chromophore in signal transduction is discussed.

Learning from hydrogenases: location of a proton pump and of a second FMN in bovine NADH--ubiquinone oxidoreductase (Complex I).

Hydrogenases have clear evolutionary links to the much more complex NADH-ubiquinone oxidoreductases (Complex I). Certain membrane-bound [NiFe]-hydrogenases presumably pump protons. From a detailed comparison of hydrogenases and Complex I, it is concluded here that the TYKY subunit in these enzymes is a special 2[4Fe-4S] ferredoxin, which functions as the electrical driving unit for a proton pump. The comparison further revealed that the flavodoxin fold from [NiFe]-hydrogenases is presumably conserved in the PSST subunit of Complex I. It is proposed that bovine Complex I and the soluble NAD(+)-reducing hydrogenase from Ralstonia eutropha each contain a second FMN group.

Structural examination of the nickel site in chromatium vinosum hydrogenase: redox state oscillations and structural changes accompanying reductive activation and CO binding.

An X-ray absorption spectroscopic study of structural changes occurring at the Ni site of Chromatium vinosum hydrogenase during reductive activation, CO binding, and photolysis is presented. Structural details of the Ni sites for the ready silent intermediate state, SI(r), and the carbon monoxide complex, SI-CO, are presented for the first time in any hydrogenase. Analysis of nickel K-edge energy shifts in redox-related samples reveals that reductive activation is accompanied by an oscillation in the electron density of the Ni site involving formally Ni(III) and Ni(II), where all the EPR-active states (forms A, B, and C) are formally Ni(III), and the EPR-silent states are formally Ni(II). Analysis of XANES shows that the Ni site undergoes changes in the coordination number and geometry that are consistent with five-coordinate Ni sites in forms A, B, and SI(u); distorted four-coordinate sites in SI(r) and R; and a six-coordinate Ni site in form C. EXAFS analysis reveals that the loss of a short Ni-O bond accounts for the change in coordination number from five to four that accompanies formation of SI(r). A shortening of the Ni-Fe distance from 2.85(5) A in form B to 2.60(5) A also occurs at the SI level and is thus associated with the loss of the bridging O-donor ligand in the active site. Multiple-scattering analysis of the EXAFS data for the SI-CO complex reveals the presence of Ni-CO ligation, where the CO is bound in a linear fashion appropriate for a terminal ligand. The putative role of form C in binding H(2) or H(-) was examined by comparing the XAS data from form C with that of its photoproduct, form L. The data rule out the suggestion that the increase in charge density on the NiFe active site that accompanies the photoprocess results in a two-electron reduction of the Ni site [Ni(III) --> Ni(I)] [Happe, R. P., Roseboom, W., and Albracht, S. P. J. (1999) Eur. J. Biochem. 259, 602-608]; only subtle structural differences between the Ni sites were observed.

Unusual FTIR and EPR properties of the H2-activating site of the cytoplasmic NAD-reducing hydrogenase from Ralstonia eutropha.

Soluble NAD-reducing [NiFe]-hydrogenase (SH) from Ralstonia eutropha (formerly Alcaligenes eutrophus) has an infrared spectrum with one strong band at 1956 cm(-1) and four weak bands at 2098, 2088, 2081 and 2071 cm(-1) in the 2150-1850 cm(-1) spectral region. Other [NiFe]-hydrogenases only show one strong and two weak bands in this region, attributable to the NiFe(CN)2(CO) active site. The position of these three bands is highly sensitive to redox changes of the active site. In contrast, reduction of the SH resulted in a shift to lower frequencies of the 2098 cm(-1) band only. These and other properties prompted us to propose the presence of a Ni(CN)Fe(CN)3(CO) active site.

Orientation-selected ENDOR of the active center in Chromatium vinosum [NiFe] hydrogenase in the oxidized "ready" state.

Electron nuclear double resonance (ENDOR) was applied to study the active site of the oxidized "ready" state, Ni(r), in the [NiFe] hydrogenase of Chromatium vinosum. The magnetic field dependence of the EPR was used to select specific subsets of molecules contributing to the ENDOR response by stepping through the EPR envelope. Three hyperfine couplings could be clearly followed over the complete field range. Two protons, H1 and H2, display a very similar large isotropic coupling of 12.5 and 12.6 MHz, respectively. Their dipolar coupling is small (2.1 and 1.4 MHz, respectively). A third proton, H3, exhibits a small isotropic coupling of 0.5 MHz and a larger anisotropic contribution of 3.5 MHz. Based on a comparison with structural data obtained from X-ray crystallography of single crystals of hydrogenases from Desulfovibrio gigas and D. vulgaris and the known g-tensor orientation of Ni(r), an assignment of the 1H hyperfine couplings could be achieved. H1 and H2 were assigned to the beta-CH2 protons of the bridging cysteine Cys533 and H3 could belong to a beta-CH2 proton of Cys68 or to a protonated cysteine (-SH) of Cys68 or Cys530.

Catalytic electron transport in Chromatium vinosum [NiFe]-hydrogenase: application of voltammetry in detecting redox-active centers and establishing that hydrogen oxidation is very fast even at potentials close to the reversible H+/H2 value.

The nickel-iron hydrogenase from Chromatium vinosum adsorbs at a pyrolytic graphite edge-plane (PGE) electrode and catalyzes rapid interconversion of H(+)((aq)) and H(2) at potentials expected for the half-cell reaction 2H(+) right arrow over left arrow H(2), i.e., without the need for overpotentials. The voltammetry mirrors characteristics determined by conventional methods, while affording the capabilities for exquisite control and measurement of potential-dependent activities and substrate-product mass transport. Oxidation of H(2) is extremely rapid; at 10% partial pressure H(2), mass transport control persists even at the highest electrode rotation rates. The turnover number for H(2) oxidation lies in the range of 1500-9000 s(-)(1) at 30 degrees C (pH 5-8), which is significantly higher than that observed using methylene blue as the electron acceptor. By contrast, proton reduction is slower and controlled by processes occurring in the enzyme. Carbon monoxide, which binds reversibly to the NiFe site in the active form, inhibits electrocatalysis and allows improved definition of signals that can be attributed to the reversible (non-turnover) oxidation and reduction of redox centers. One signal, at -30 mV vs SHE (pH 7.0, 30 degrees C), is assigned to the [3Fe-4S](+/0) cluster on the basis of potentiometric measurements. The second, at -301 mV and having a 1. 5-2.5-fold greater amplitude, is tentatively assigned to the two [4Fe-4S](2+/+) clusters with similar reduction potentials. No other redox couples are observed, suggesting that these two sets of centers are the only ones in CO-inhibited hydrogenase capable of undergoing simple rapid cycling of their redox states. With the buried NiFe active site very unlikely to undergo direct electron exchange with the electrode, at least one and more likely each of the three iron-sulfur clusters must serve as relay sites. The fact that H(2) oxidation is rapid even at potentials nearly 300 mV more negative than the reduction potential of the [3Fe-4S](+/0) cluster shows that its singularly high equilibrium reduction potential does not compromise catalytic efficiency.

Pre-steady-state kinetics of the reactions of [NiFe]-hydrogenase from Chromatium vinosum with H2 and CO.

Results are presented of the first rapid-mixing/rapid-freezing studies with a [NiFe]-hydrogenase. The enzyme from Chromatium vinosum was used. In particular the reactions of active enzyme with H2 and CO were monitored. The conversion from fully reduced, active hydrogenase (Nia-SR state) to the Nia-C* state was completed in less than 8 ms, a rate consistent with the H2-evolution activity of the enzyme. The reaction of CO with fully reduced enzyme was followed from 8 to 200 ms. The Nia-SR state did not react with CO. It was discovered, contrary to expectations, that the Nia-C* state did not react with CO when reactions were performed in the dark. When H2 was replaced by CO, a Nia-C* EPR signal appeared within 11 ms; this was also the case when H2 was replaced by Ar. With CO, however, the Nia-C* state decayed within 40 ms, due to the generation of the Nia-S.CO state (the EPR-silent state of the enzyme with bound CO). The Nia-C* state, induced after 11 ms by replacing H2 by CO in the dark, could be converted, in the frozen enzyme, into the EPR-detectable state with CO bound to nickel (Nia*.CO) by illumination at 30 K (evoking the Nia-L* state), followed by dark adaptation at 200 K. This can be explained by assuming that the Nia-C* state represents a formally trivalent state of nickel, which is unable to bind CO, whereas nickel in the Nia-L* and the Nia*.CO states is formally monovalent.

Carbon monoxide and cyanide as intrinsic ligands to iron in the active site of [NiFe]-hydrogenases. NiFe(CN)2CO, Biology's way to activate H2.

Infrared-spectroscopic studies on the [NiFe]-hydrogenase of Chromatium vinosum-enriched in 15N or 13C, as well as chemical analyses, show that this enzyme contains three non-exchangeable, intrinsic, diatomic molecules as ligands to the active site, one carbon monoxide molecule and two cyanide groups. The results form an explanation for the three non-protein ligands to iron detected in the crystal structure of the Desulfovibrio gigas hydrogenase (Volbeda, A., Garcin, E., Piras, C., De Lacey, A. I., Fernandez, V. M., Hatchikian, E. C., Frey, M., and Fontecilla-Camps, J. C. (1996) J. Am. Chem. Soc. 118, 12989-12996) and for the low spin character of the lone ferrous iron ion observed with Mössbauer spectroscopy (Surerus, K. K., Chen, M., Van der Zwaan, W., Rusnak, F. M., Kolk, M. , Duin, E. C., Albracht, S. P. J., and Münck, E. (1994) Biochemistry 33, 4980-4993). The results do not support the notion, based upon studies of Desulfovibrio vulgaris [NiFe]-hydrogenase (Higuchi, Y., Yagi, T., and Noritake, Y. (1997) Structure 5, 1671-1680), that SO is a ligand to the active site. The occurrence of both cyanide and carbon monoxide as intrinsic constituents of a prosthetic group is unprecedented in biology.

Characterization of the active site of a hydrogen sensor from Alcaligenes eutrophus.

A third hydrogenase was recently identified in the proteobacterium Alcaligenes eutrophus as a constituent of a novel H2-sensing multicomponent regulatory system. This regulatory hydrogenase (RH) has been overexpressed in cells deficient in both the NAD+-reducing [NiFe]-hydrogenase and the membrane-bound [NiFe]-hydrogenase. EPR, FTIR and activity studies of membrane-free extracts revealed that the RH has an active site much like that of standard [NiFe]-hydrogenases, i.e. a Ni-Fe site with two CN- groups and one CO molecule. Its catalytic power is low, but the RH is always active, insensitive to oxygen, and occurs in only two redox states.

Comparison of energization of complex I in membrane particles from Paracoccus denitrificans and bovine heart mitochondria.

The results of preliminary studies of the effects of energization on the catalytic and EPR properties of complex I in tightly coupled membrane vesicles of Paracoccus denitrificans (SPP) are presented. They are compared to those observed in submitochondrial particles from bovine heart (SMP). All signs of energization of complex I detected by EPR in SMP (uncoupler-sensitive splitting of the gz lines of the clusters 2 and a broadening of their gxy lines, a fast-relaxing, piericidin-sensitive ubiquinone-radical signal, and a broad signal around g = 1.94) were also observed with the bacterial enzyme. There were some prominent differences, though. The signal of the fast-relaxing radicals could be evoked both in the presence or absence of reduced clusters 2, suggesting that enhancement of its spin-relaxation rate is caused by coupling to another paramagnet. The signal was hardly affected by the presence of gramicidin. The slow-relaxing radical signal did not disappear upon anaerobiosis, but was detectable for at least another 30 s. The fast-relaxing signal vanished immediately upon anaerobiosis. The activity of the bacterial enzyme during oxidation of NADH by oxygen or reduction of NAD induced by succinate oxidation, was 5-6 times higher than that of the mitochondrial enzyme. Unlike the mitochondrial enzyme, the bacterial enzyme was not inactivated by incubation at 35 degrees C. The spin concentration of the NADH-reducible [2Fe-2S] cluster (1b) was half that of the clusters 2, indicating no difference with the mitochondrial enzyme.

Distal genes of the nuo operon of Rhodobacter capsulatus equivalent to the mitochondrial ND subunits are all essential for the biogenesis of the respiratory NADH-ubiquinone oxidoreductase.

Seven out of the 13 proteins encoded by the mitochondrial genome of mammals (peptides ND1 to ND6 plus ND4L) are subunits of the respiratory NADH-ubiquinone oxidoreductase (complex I). The function of these ND subunits is still poorly understood. We have used the NADH-ubiquinone oxidoreductase of Rhodobacter capsulatus as a model for the study of the function of these proteins. In this bacterium, the 14 genes encoding the NADH-ubiquinone oxidoreductase are clustered in the nuo operon. We report here on the biochemical and spectroscopic characterization of mutants individually disrupted in five nuo genes, equivalent to mitochondrial genes nd1, nd2, nd5, nd6 and nd4L. Disruption of any of these genes in R. capsulatus leads to the suppression of NADH dehydrogenase activity at the level of the bacterial membranes and to the disappearance of complex I-associated iron-sulphur clusters. Individual NUO subunits can still be immunodetected in the membranes of these mutants, but they do not form a functional subcomplex. In contrast to these observations, disruption of two ORFs (orf6 and orf7), also present in the distal part of the nuo operon, does not suppress NADH dehydrogenase activity or complex I-associated EPR signals, thus demonstrating that these ORFs are not essential for the biosynthesis of complex I.

Identification of the 4-glutamyl radical as an intermediate in the carbon skeleton rearrangement catalyzed by coenzyme B12-dependent glutamate mutase from Clostridium cochlearium.

A series of 2H- and 13C-labeled glutamates were used as substrates for coenzyme B12-dependent glutamate mutase, which equilibrates (S)-glutamate with (2S,3S)-3-methylaspartate. These compounds contained the isotopes at C-2, C-3, or C-4 of the carbon chain: [2-2H], [3,3-2H2], [4,4-2H2], [2,3,3,4,4-2H5], [2-13C], [3-13C], and [4-13C]glutamate. Each reaction was monitored by electron paramagnetic resonance (EPR) spectroscopy and revealed a similar signal characterized by g'xy = 2.1, g'z = 1.985, and A' = 5.0 mT. The interpretation of the spectral data was aided by simulations which gave close agreement with experiment. This approach underpinned the idea of the formation of a radical pair, consisting of cob(II)alamin interacting with an organic radical at a distance of 6.6 +/- 0.9 A. Comparison of the hyperfine couplings observed with unlabeled glutamate with those from the labeled glutamates enabled a principal contributor to the radical pair to be identified as the 4-glutamyl radical. These findings support the currently accepted mechanism for the glutamate mutase reaction, i.e., the process is initiated through hydrogen atom abstraction from C-4 of glutamate by the 5'-deoxyadenosyl radical, which is derived by homolysis of the Co-C sigma-bond of coenzyme B12.

A low-spin iron with CN and CO as intrinsic ligands forms the core of the active site in [Fe]-hydrogenases.

In this report the first high-quality infrared spectra of [Fe]-hydrogenase are presented. Analyses of these spectra obtained under a variety of redox conditions strongly indicate that [Fe]-hydrogenases contain a low-spin Fe ion in the active site with one CN- group and one CO molecule as intrinsic, non-protein ligands. When in the ferric state, the presence of such an ion can explain the enigmatic EPR properties (the rhombic 2.10 signal) of the active, oxidised enzyme. To account for other, well-characterised properties of the active site, we propose that the active site of [Fe]-hydrogenases consists of this low-spin Fe ion bound to an unusual [4Fe-4S] cluster via bridges with sulphur atoms.

Changes in the electronic structure around Ni in oxidized and reduced selenium-containing hydrogenases from Methanococcus voltae.

The selenium-containing F420-reducing hydrogenase from Methanococcus voltae was anaerobically purified to a specific hydrogen-uptake activity of 350 U/mg protein as determined with the natural electron acceptor. The concentrated enzyme was used for EPR-spectroscopic investigations. As isolated, the enzyme showed an EPR spectrum with g(xyz) values of 2.21, 2.15 and 2.01. Illumination of such samples at low temperatures led to an EPR spectrum with g(xyz) values of 2.05, 2.11 and 2.29. These spectra are typical for [NiFe]hydrogenases in the active state. Spectra of samples enriched in 77Se showed a hyperfine interaction between the unpaired spin of the nickel ion and the nuclear spin of one 77Se atom before and after illumination. A 90 degree flip of the electronic z-axis is proposed to explain the hyperfine interaction in both states. This has been demonstrated previously only for the F420-non-reducing hydrogenase from M. voltae, where the selenium atom is present as a selenocysteine residue on an unusually small separate subunit [Sorgenfrei, O., Klein, A. & Albracht, S. P. J. (1993) FEBS Lett. 332, 291-297]. The results demonstrate that the three-dimensional structures of the active sites in the selenium-containing F420-reducing and F420-non-reducing hydrogenases from M. voltae are highly similar and hence are not influenced by the unusual subunit structure of the latter enzyme. Oxidized samples containing either natural selenium or 77Se were prepared from the F420-reducing and the selenium-containing F420-non-reducing hydrogenase. Both enzymes exhibited EPR spectra typical for [NiFe]hydrogenases in the inactive 'ready' state. In contrast to the reduced form, no splitting of the nickel-derived signal due to the nuclear spin of 77Se was observed in the oxidized state, indicating that the electronic z-axis is perpendicular to the Ni-Se direction.

Characterization of the respiratory chain from cultured Crithidia fasciculata.

Mitochondrial mRNAs encoding subunits of respiratory-chain complexes in kinetoplastids are post-transcriptionally edited by uridine insertion and deletion. In order to identify the proteins encoded by these mRNAs, we have analyzed respiratory-chain complexes from cultured cells of Crithidia fasciculata with the aid of 2D polyacrylamide gel electrophoresis (PAGE). The subunit composition of F0F1-ATPase (complex V), identified on the basis of its activity as an oligomycin-sensitive ATPase, is similar to that of bovine mitochondrial F0F1-ATPase. Amino acid sequence analysis, combined with binding studies using dicyclohexyldiimide and azido ATP allowed the identification of two F0 subunits (b and c) and all of the F1 subunits. The F0 b subunit has a low degree of similarity to subunit b from other organisms. The F1 alpha subunit is extremely small making the beta subunit the largest F1 subunit. Other respiratory-chain complexes were also analyzed. Interestingly, an NADH: ubiquinone oxidoreductase (complex I) appeared to be absent, as judged by electron paramagnetic resonance (EPR), enzyme activity and 2D PAGE analysis. Cytochrome c oxidase (complex IV) displayed a subunit pattern identical to that reported for the purified enzyme, whereas cytochrome c reductase (complex III) appeared to contain two extra subunits. A putative complex II was also identified. The amino acid sequences of the subunits of these complexes also show a very low degree of similarity (if any) to the corresponding sequences in other organisms. Remarkably, peptide sequences derived from mitochondrially encoded subunits were not found in spite of the fact that sequences were obtained of virtually all subunits of complex III, IV and V.

Bovine-heart NADH:ubiquinone oxidoreductase is a monomer with 8 Fe-S clusters and 2 FMN groups.

The availability of the amino-acid sequences of a number of mitochondrial and bacterial NADH:ubiquinone oxidoreductases (Complex I), the sequence similarities of five of the essential subunits of Complex I with subunits of [NiFe]hydrogenases and [Fe]hydrogenases, as well as some long-standing controversies about the precise EPR properties and stoichiometries of the iron-sulfur clusters in Complex I have led us to propose a new structural and functional model for this complicated enzyme. The functional unit is a monomer comprising 8 different Fe-S clusters and 2 FMN molecules as prosthetic groups. The electron-input pathway, as well as part of the electron-transfer components, seem largely inherited from bacterial NAD(+)-reducing hydrogenases. The essential electron-transfer components of the electron-output pathway are located in the TYKY subunit. This subunit is proposed to hold both iron-sulfur clusters 2 and to render the enzyme the ability to perform coupled electron transfer. Based on earlier observed similarities (Albracht. S.P.J. (1993) Biochim. Biophys. Acta 1144, 221-224) of the 49 kDa subunit and the PSST subunit with, respectively, the large and small subunits of [NiFe]hydrogenases, it is proposed that the 49 kDa/PSST subunit couple provides Complex I with an ancient proton-transfer pathway.