RESUMO
This paper presents a new assay to determine the activity of the lysosomal enzyme α-N-acetylgalactosaminidase (Naga, EC 3.2.1.49) in human serum. It is based on the use of a new chromogenic substrate, DNP-α-GalNAc (2,4-dinitrophenyl-N-acetyl-α-D-galactosaminide) and is performed at pH 4.3 and 37 °C. This allows continuous monitoring of the absorbance of the released DNP. The assay can be performed with a standard spectrophotometer. Compared to established methods using an endpoint assay with MU-α-GalNAc (4-methylumbelliferyl-GalNAc), the present method gives a ca. 3-fold higher specific activity, while only one tenth of the serum concentration in the assay is required. Hence, the assay is at least 30-fold more sensitive than that with MU-α-GalNAc. The pH dependence of the reaction with DNP-α-GalNAc in the pH 3.5 to 6.5 region, while using 4% serum in the assay, shows only one peak around pH 4. This pH optimum is similar to that reported with MU-α-GalNAc. In the accompanying paper (Albracht and Van Pelt (2017) Multiple exo-glycosidases in human serum as detected with the substrate DNP-α-GalNAc. II. Three α-N-acetylgalactosaminidase-like activities in the pH 5 to 8 region. Biochim. Biophys. Acta 159 (2017) Part I and II), the method is used to show that, under special assay conditions, three more Naga-like activities can be uncovered in human serum.
RESUMO
With the substrate DNP-α-GalNAc (2,4-dinitrophenyl-N-acetyl-α-d-galactosaminide) three α-N-acetylgalactosaminidase-like activities could be distinguished in serum, in addition to the classical lysosomal enzyme (Naga, EC 3.2.1.49, pH optimum at 4). Two activities had optima in the pH 5 to 6 region and one peaked around pH 8. Like the Naga activity at pH 4, the activity at pH 8 was detectable under standard assay conditions. However, the two activities in the pH 5 to 6 range were not readily apparent in such assays. They could be unmasked as separate activities only when low serum concentrations were used. Addition of 1% saturated ammonium sulphate to the assay medium stimulated these activities. All activities in the pH 5 to 8 range decreased with increasing serum concentration in the assay, suggesting the presence of endogenous inhibitors. The activities between pH 5 and 6 might be similar to an activity described in 1996, which was considerably elevated in serum of patients with great variety of cancers (N. Yamamoto, V.R. Naraparaju, and S.O. Asbell (1996). Deglycosylation of serum vitamin D3-binding protein leads to immunosuppression in cancer patients. Cancer Res. 56, 2827-2811).
RESUMO
Mammalian NADH:ubiquinone oxidoreductase (Complex I) in the mitochondrial inner membrane catalyzes the oxidation of NADH in the matrix. Excess NADH reduces nine of the ten prosthetic groups of the enzyme in bovine-heart submitochondrial particles with a rate of at least 3,300 s⻹. This results in an overall NADHâO2 rate of ca. 150 s⻹. It has long been known that the bovine enzyme also has a specific reaction site for NADPH. At neutral pH excess NADPH reduces only three to four of the prosthetic groups in Complex I with a rate of 40 s⻹ at 22 °C. The reducing equivalents remain essentially locked in the enzyme because the overall NADPHâO2 rate (1.4 s⻹) is negligible. The physiological significance of the reaction with NADPH is still unclear. A number of recent developments has revived our thinking about this enigma. We hypothesize that Complex I and the Δp-driven nicotinamide nucleotide transhydrogenase (Nnt) co-operate in an energy-dependent attenuation of the hydrogen-peroxide generation by Complex I. This co-operation is thought to be mediated by the NADPH/NADP⺠ratio in the vicinity of the NADPH site of Complex I. It is proposed that the specific H2O2 production by Complex I, and the attenuation of it, is of importance for apoptosis, autophagy and the survival mechanism of a number of cancers. Verification of this hypothesis may contribute to a better understanding of the regulation of these processes.
Assuntos
Complexo I de Transporte de Elétrons/metabolismo , Peróxido de Hidrogênio/metabolismo , NADP Trans-Hidrogenases/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias/enzimologia , Animais , Humanos , MamíferosRESUMO
The first purification of bovine NADH:ubiquinone oxidoreductase (Complex I) was reported nearly half a century ago (Hatefi et al. J Biol Chem 237:1676-1680, 1962). The pathway of electron-transfer through the enzyme is still under debate. A major obstacle is the assignment of EPR signals to the individual iron-sulfur clusters in the subunits. The preceding paper described a working model based on the kinetics with NADPH. This model is at variance with current views in the field. The present paper provides a critical overview on the possible causes for the discrepancies. It is concluded that the stability of all purified preparations described thus far, including Hatefi's Complex I, is compromised due to removal of the enzyme from the protective membrane environment. In addition, most preparations described during the last two decades are purified by methods involving synthetic detergents and column chromatography. This results in delipidation, loss of endogenous quinones and loss of reactions with (artificial) quinones in a rotenone-sensitive way. The Fe:FMN ratio's indicate that FMN-a is absent, but that all Fe-S clusters may be present. In contrast to the situation in bovine SMP and Hatefi's Complex I, three of the six expected [4Fe-4S] clusters are not detected in EPR spectra. Qualitatively, the overall EPR lineshape of the remaining three cubane signals may seem similar to that of Hatefi's Complex I, but quantitatively it is not. It is further proposed that point mutations in any of the TYKY, PSST, 49-kDa or 30-kDa subunits, considered to make up the delicate structural heart of Complex I, may have unpredictable effects on any of the other subunits of this quartet. The fact that most point mutations led to inactive enzymes makes a correct interpretation of such mutations even more ambiguous. In none of the Complex-I-containing membrane preparations from non-bovine origin, the pH dependencies of the NAD(P)H-->O(2) reactions and the pH-dependent reduction kinetics of the Fe-S clusters with NADPH have been determined. This excludes a proper discussion on the absence or presence of FMN-a in native Complex I from other organisms.
Assuntos
Complexo I de Transporte de Elétrons/química , Complexo I de Transporte de Elétrons/metabolismo , Mitocôndrias Cardíacas/enzimologia , NADP/metabolismo , Animais , Bovinos , Espectroscopia de Ressonância de Spin Eletrônica , Transporte de Elétrons , Humanos , Mitocôndrias Cardíacas/metabolismo , NAD/metabolismoRESUMO
Bovine NADH:ubiquinone oxidoreductase (Complex I) is the first complex in the mitochondrial respiratory chain. It has long been assumed that it contained only one FMN group. However, as demonstrated in 2003, the intact enzyme contains two FMN groups. The second FMN was proposed to be located in a conserved flavodoxin fold predicted to be present in the PSST subunit. The long-known reaction of Complex I with NADPH differs in many aspects from that with NADH. It was proposed that the second flavin group was specifically involved in the reaction with NADPH. The X-ray structure of the hydrophilic domain of Complex I from Thermus thermophilus (Sazanov and Hinchliffe 2006, Science 311, 1430-1436) disclosed the positions of all redox groups of that enzyme and of the subunits holding them. The PSST subunit indeed contains the predicted flavodoxin fold although it did not contain FMN. Inspired by this structure, the present paper describes a re-evaluation of the enigmatic reactions of the bovine enzyme with NADPH. Published data, as well as new freeze-quench kinetic data presented here, are incompatible with the general opinion that NADPH and NADH react at the same site. Instead, it is proposed that these pyridine nucleotides react at opposite ends of the 90 A long chain of prosthetic groups in Complex I. Ubiquinone is proposed to react with the Fe-S clusters in the TYKY subunit deep inside the hydrophilic domain. A new model for electron transfer in Complex I is proposed. In the accompanying paper this model is compared with the one advocated in current literature.
Assuntos
Complexo I de Transporte de Elétrons/metabolismo , Mitocôndrias Cardíacas/enzimologia , NADP/metabolismo , Animais , Bovinos , Espectroscopia de Ressonância de Spin Eletrônica , Complexo I de Transporte de Elétrons/química , Humanos , Concentração de Íons de Hidrogênio , Mitocôndrias Cardíacas/metabolismo , Modelos Moleculares , NAD/metabolismo , Oxirredução , Oxigênio/metabolismoRESUMO
Hydrogenases are enzymes which catalyze the reversible cleavage of molecular hydrogen into protons and electrons. In [FeFe] hydrogenases the active center is a 6Fe6S cluster, referred to as the "H-cluster." It consists of the redox-active binuclear subcluster ([2Fe](H)) coordinated by CN(-) and CO ligands and the cubane-like [4Fe-4S](H) subcluster which is connected to the protein via Cys ligands. One of these Cys ligands bridges to the [2Fe](H) subcluster. The CO-inhibited form of [FeFe] hydrogenase isolated from Desulfovibrio desulfuricans was studied using advanced EPR methods. In the H(ox)-CO state the open coordination site at the [2Fe](H) subcluster is blocked by extrinsic CO, giving rise to an EPR-active S = 1/2 species. The CO inhibited state was prepared with (13)CO and illuminated under white light at 273 K. In this case scrambling of the CO ligands occurs. Three (13)C hyperfine couplings of 17.1, 7.4, and 3.8 MHz (isotropic part) were observed and assigned to (13)CO at the extrinsic, the bridging, and the terminal CO-ligand positions of the distal iron, respectively. No (13)CO exchange of the CO ligand to the proximal iron was observed. The hyperfine interactions detected indicate a rather large distribution of the spin density over the terminal and bridging CO ligands attached to the distal iron. Furthermore, (14)N nuclear spin interactions were measured. On the basis of the observed (14)N hyperfine couplings, which result from the CN(-) ligands of the [2Fe](H) subcluster, it has been concluded that there is very little unpaired spin density on the cyanides of the binuclear subcluster.
Assuntos
Monóxido de Carbono/química , Desulfovibrio desulfuricans/enzimologia , Hidrogênio/química , Hidrogenase/química , Proteínas Ferro-Enxofre/química , Isótopos de Carbono , Simulação por Computador , Cianetos/química , Desulfovibrio desulfuricans/crescimento & desenvolvimento , Espectroscopia de Ressonância de Spin Eletrônica , Ligantes , Conformação Molecular , Nitrogênio/química , Padrões de ReferênciaRESUMO
We report on the use of polymyxin (PM), a cyclic cationic lipodecapeptide, as an electrode modifier for studying protein film voltammetry (PFV) on Au and single-walled carbon nanotube (SWNT) electrodes. Pretreating the electrodes with PM allows for the subsequent immobilization of an active submonolayer of [NiFe]-hydrogenase from Allochromatium vinosum ( Av H2ase). Probed by cyclic voltammetry (CV), the adsorbed enzyme exhibits characteristic electrocatalytic behavior that is stable for several hours under continuous potential cycling. An unexpected feature of the immobilization procedure is that the presence of chloride ions is a prerequisite for obtaining electrocatalytic activity. Atomic force microscopy (AFM) relates the observed catalytic activity to enzymatic adsorption at the PM/Au(111) surface, and a combination of concentration-dependent CV and AFM is used to investigate the interaction between the enzyme and the PM layer.
Assuntos
Proteínas de Bactérias/química , Gammaproteobacteria/enzimologia , Ouro/química , Hidrogenase/química , Nanotubos de Carbono/química , Polimixinas/química , Cloretos/química , Eletroquímica , Eletrodos , Enzimas Imobilizadas/químicaRESUMO
We have scaled down electrochemical assays of redox-active enzymes enabling us to study small numbers of molecules. Our approach is based on lithographically fabricated Au nanoelectrodes with dimensions down to ca. 70 x 70 nm(2). We first present a detailed characterization of the electrodes using a combination of scanning electron microscopy, cyclic voltammetry, and finite-element modeling. We then demonstrate the viability of the approach by focusing on the highly active [NiFe]-hydrogenase from Allochromatium vinosum immobilized on polymyxin-pretreated Au. Using this system, we successfully demonstrate a distinct catalytic response from less than 50 enzyme molecules. These results strongly suggest the feasibility of using bioelectrochemistry as a new tool for studying redox enzymes at the single-molecule level.
Assuntos
Chromatiaceae/enzimologia , Eletroquímica/métodos , Hidrogenase/química , Eletrodos , Enzimas Imobilizadas/química , Microscopia Eletrônica de Varredura , Modelos MolecularesRESUMO
The active site of the (57)Fe-enriched [FeFe]-hydrogenase (i.e., the "H-cluster") from Desulfovibrio desulfuricans has been examined using advanced pulse EPR methods at X- and Q-band frequencies. For both the active oxidized state (H(ox)) and the CO inhibited form (H(ox)-CO) all six (57)Fe hyperfine couplings were detected. The analysis shows that the apparent spin density extends over the whole H-cluster. The investigations revealed different hyperfine couplings of all six (57)Fe nuclei in the H-cluster of the H(ox)-CO state. Four large 57Fe hyperfine couplings in the range 20-40 MHz were found (using pulse ENDOR and TRIPLE methods) and were assigned to the [4Fe-4S](H) (cubane) subcluster. Two weak (57)Fe hyperfine couplings below 5 MHz were identified using Q-band HYSCORE spectroscopy and were assigned to the [2Fe](H) subcluster. For the H(ox) state only two different 57Fe hyperfine couplings in the range 10-13 MHz were detected using pulse ENDOR. An (57)Fe line broadening analysis of the X-band CW EPR spectrum indicated, however, that all six (57)Fe nuclei in the H-cluster are contributing to the hyperfine pattern. It is concluded that in both states the binuclear subcluster [2Fe](H) assumes a [Fe(I)Fe(II)] redox configuration where the paramagnetic Fe(I) atom is attached to the [4Fe-4S](H) subcluster. The (57)Fe hyperfine interactions of the formally diamagnetic [4Fe-4S](H) are due to an exchange interaction between the two subclusters as has been discussed earlier by Popescu and Münck [Popescu, C.V.; Münck, E., J. Am. Chem. Soc. 1999, 121, 7877-7884]. This exchange coupling is strongly enhanced by binding of the extrinsic CO ligand. Binding of the dihydrogen substrate may induce a similar effect, and it is therefore proposed that the observed modulation of the electronic structure by the changing ligand surrounding plays an important role in the catalytic mechanism of [FeFe]-hydrogenase.
Assuntos
Proteínas de Bactérias/química , Desulfovibrio desulfuricans/enzimologia , Hidrogenase/química , Proteínas Ferro-Enxofre/química , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Monóxido de Carbono/química , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Elétrons , Hidrogenase/metabolismo , Proteínas Ferro-Enxofre/metabolismo , Modelos Moleculares , Oxirredução , Conformação ProteicaRESUMO
Electron paramagnetic resonance (EPR) and Fourier transform IR studies on the soluble hydrogenase from Acidithiobacillus ferrooxidans are presented. In addition, detailed sequence analyses of the two subunits of the enzyme have been performed. They show that the enzyme belongs to a group of uptake [NiFe] hydrogenases typical for Cyanobacteria. The sequences have also a close relationship to those of the H(2)-sensor proteins, but clearly differ from those of standard [NiFe] hydrogenases. It is concluded that the structure of the catalytic centre is similar, but not identical, to that of known [NiFe] hydrogenases. The active site in the majority of oxidized enzyme molecules, 97% in cells and more than 50% in the purified enzyme, is EPR-silent. Upon contact with H(2) these sites remain EPR-silent and show only a limited IR response. Oxidized enzyme molecules with an EPR-detectable active site show a Ni(r)*-like EPR signal which is light-sensitive at cryogenic temperatures. This is a novelty in the field of [NiFe] hydrogenases. Reaction with H(2) converts these active sites to the well-known Ni(a)-C* state. Illumination below 160 K transforms this state into the Ni(a)-L* state. The reversal, in the dark at 200 K, proceeds via an intermediate Ni EPR signal only observed with the H(2)-sensor protein from Ralstonia eutropha. The EPR-silent active sites in as-isolated and H(2)-treated enzyme are also light-sensitive as observed by IR spectra at cryogenic temperatures. The possible origin of the light sensitivity is discussed. This study represents the first spectral characterization of an enzyme of the group of cyanobacterial uptake hydrogenases.
Assuntos
Acidithiobacillus/enzimologia , Hidrogenase/química , Hidrogenase/isolamento & purificação , Sequência de Aminoácidos , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Hidrogênio/química , Hidrogênio/metabolismo , Ferro/química , Proteínas Ferro-Enxofre , Cinética , Luz , Níquel/química , Oxirredução , Espectroscopia de Infravermelho com Transformada de Fourier/métodosRESUMO
A soluble hydrogenase from Allochromatium vinosum was purified. It consisted of a large (M (r) = 52 kDa) and a small (M (r) = 23 kDa) subunit. The genes encoding for both subunits were identified. They belong to an open reading frame where they are preceded by three more genes. A DNA fragment containing all five genes was cloned and sequenced. The deduced amino acid sequences of the products characterized the complex as a member of the HoxEFUYH type of [NiFe] hydrogenases. Detailed sequence analyses revealed binding sites for eight Fe-S clusters, three [2Fe-2S] clusters and five [4Fe-4S] clusters, six of which are also present in homologous subunits of [FeFe] hydrogenases and NADH:ubiquione oxidoreductases (complex I). This makes the HoxEFUYH type of hydrogenases the one that is evolutionary closest to complex I. The relative positions of six of the potential Fe-S clusters are predicted on the basis of the X-ray structures of the Clostridium pasteurianum [FeFe] hydrogenase I and the hydrophilic domain of complex I from Thermus thermophilus. Although the HoxF subunit contains binding sites for flavin mononucleotide and NAD(H), cell-free extracts of A. vinosum did not catalyse a H(2)-dependent reduction of NAD(+). Only the hydrogenase module (HoxYH) could be purified. Its electron paramagnetic resonance (EPR) and IR spectral properties showed the presence of a Ni-Fe active site and a [4Fe-4S] cluster. Its activity was sensitive to carbon monoxide. No EPR signals from a light-sensitive Ni(a)-C* state could be observed. This study presents the first IR spectroscopic data on the HoxYH module of a HoxEFUYH type of [NiFe] hydrogenase.
Assuntos
Chromatiaceae/enzimologia , Hidrogenase/química , Sequência de Aminoácidos , Chromatiaceae/crescimento & desenvolvimento , DNA/química , DNA/isolamento & purificação , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Ativação Enzimática , Hidrogenase/genética , Hidrogenase/isolamento & purificação , Modelos Moleculares , Dados de Sequência Molecular , Sensibilidade e Especificidade , Espectrofotometria Infravermelho , Fatores de TempoRESUMO
Infrared (IR) spectra in combination with chemical analyses have recently shown that the active Ni-Fe site of the soluble NAD(+)-reducing [NiFe]-hydrogenase from Ralstonia eutropha contains four cyanide groups and one carbon monoxide as ligands. Experiments presented here confirm this result, but show that a variable percentage of enzyme molecules loses one or two of the cyanide ligands from the active site during routine purification. For this reason the redox conditions during the purification have been optimized yielding hexameric enzyme preparations (HoxFUYHI(2)) with aerobic specific H(2)-NAD(+) activities of 150-185 mumol/min/mg of protein (up to 200% of the highest activity previously reported in the literature). The preparations were highly homogeneous in terms of the active site composition and showed superior IR spectra. IR spectro-electrochemical studies were consistent with the hypothesis that only reoxidation of the reduced enzyme with dioxygen leads to the inactive state, where it is believed that a peroxide group is bound to nickel. Electron paramagnetic resonance experiments showed that the radical signal from the NADH-reduced enzyme derives from the semiquinone form of the flavin (FMN-a) in the hydrogenase module (HoxYH dimer), but not of the flavin (FMN-b) in the NADH-dehydrogenase module (HoxFU dimer). It is further demonstrated that the hexameric enzyme remains active in the presence of NADPH and air, whereas NADH and air lead to rapid destruction of enzyme activity. It is proposed that the presence of NADPH in cells keeps the enzyme in the active state.
Assuntos
Proteínas de Bactérias , Cupriavidus necator/enzimologia , Hidrogenase , Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Estabilidade Enzimática , Hidrogenase/química , Hidrogenase/isolamento & purificação , Hidrogenase/metabolismo , Estrutura Molecular , Oxirredução , Análise EspectralRESUMO
In [FeFe]-hydrogenases, the H cluster (hydrogen-activating cluster) contains a di-iron centre ([2Fe]H subcluster, a (L)(CO)(CN)Fe(mu-RS2)(mu-CO)Fe(CysS)(CO)(CN) group) covalently attached to a cubane iron-sulphur cluster ([4Fe-4S]H subcluster). The Cys-thiol functions as the link between one iron (called Fe1) of the [2Fe]H subcluster and one iron of the cubane subcluster. The other iron in the [2Fe]H subcluster is called Fe2. The light sensitivity of the Desulfovibrio desulfuricans enzyme in a variety of states has been studied with infrared (IR) spectroscopy. The aerobic inactive enzyme (H(inact) state) and the CO-inhibited active form (H(ox)-CO state) were stable in light. Illumination of the H(ox) state led to a kind of cannibalization; in some enzyme molecules the H cluster was destroyed and the released CO was captured by the H clusters in other molecules to form the light-stable H(ox)-CO state. Illumination of active enzyme under 13CO resulted in the complete exchange of the two intrinsic COs bound to Fe2. At cryogenic temperatures, light induced the photodissociation of the extrinsic CO and the bridging CO of the enzyme in the H(ox)-CO state. Electrochemical redox titrations showed that the enzyme in the H(inact) state converts to the transition state (H(trans)) in a reversible one-electron redox step (E (m, pH 7) = -75 mV). IR spectra demonstrate that the added redox equivalent not only affects the [4Fe-4S]H subcluster, but also the di-iron centre. Enzyme in the H(trans) state reacts with extrinsic CO, which binds to Fe2. The H(trans) state converts irreversibly into the H(ox) state in a redox-dependent reaction most likely involving two electrons (E (m, pH 7) = -261 mV). These electrons do not end up on any of the six Fe atoms of the H cluster; the possible destiny of the two redox equivalents is discussed. An additional reversible one-electron redox reaction leads to the H(red) state (E (m, pH 7) = -354 mV), where both Fe atoms of the [2Fe]H subcluster have the same formal oxidation state. The possible oxidation states of Fe1 and Fe2 in the various enzyme states are discussed. Low redox potentials (below -500 mV) lead to destruction of the [2Fe]H subcluster.
Assuntos
Monóxido de Carbono/química , Desulfovibrio desulfuricans/enzimologia , Hidrogenase/química , Proteínas Ferro-Enxofre/química , Ferro/química , Fotofobia , Sítios de Ligação , Eletroquímica , Concentração de Íons de Hidrogênio , Hidrogenase/metabolismo , Proteínas Ferro-Enxofre/metabolismo , Ligantes , Luz , Oxirredução , Espectrofotometria Infravermelho/métodosRESUMO
The hydrogen-activating cluster (H cluster) in [FeFe]-hydrogenases consists of two moieties. The [2Fe]H subcluster is a (L)(CO)(CN)Fe(mu-RS2)(mu-CO)Fe(CysS)(CO)(CN) centre. The Cys-bound Fe is called Fe1, the other iron Fe2. The Cys-thiol forms a bridge to a [4Fe-4S] cluster, the [4Fe-4S]H subcluster. We report that electron paramagnetic resonance (EPR) spectra of the 57Fe-enriched enzyme from Desulfovibrio desulfuricans in the H(ox)-CO state are consistent with a magnetic hyperfine interaction of the unpaired spin with all six Fe atoms of the H cluster. In contrast to the inactive aerobic enzyme, the active enzyme is easily destroyed by light. The [2Fe]H subcluster in some enzyme molecules loses CO by photolysis, whereupon other molecules firmly bind the released CO to form the H(ox)-CO state giving rise to the so-called axial 2.06 EPR signal. Though not destroyed by light, the H(ox)-CO state is affected by it. As demonstrated in the accompanying paper [49] two of the intrinsic COs, both bound to Fe2, can be exchanged by extrinsic 13CO during illumination at 2 degrees C. We found that only one of the three 13COs, the one at the extrinsic position, gives an EPR-detectable isotropic superhyperfine interaction of 0.6 mT. At 30 K both the inhibiting extrinsic CO bound to Fe2 and one more CO can be photolysed. EPR spectra of the photolysed products are consistent with a 3d7 system of Fe with the formal oxidation state +1. The damaged enzyme shows a light-sensitive g = 5 signal which is ascribed to an S = 3/2 form of the [2Fe](H) subcluster. The light sensitivity of the enzyme explains the occurrence of the g = 5 signal and the axial 2.06 signal in published EPR spectra of nearly all preparations studied thus far.
Assuntos
Desulfovibrio desulfuricans/enzimologia , Hidrogenase/química , Proteínas Ferro-Enxofre/química , Ferro/química , Luz , Magnetismo , Sítios de Ligação , Monóxido de Carbono/química , Cisteína/química , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Hidrogenase/metabolismo , Proteínas Ferro-Enxofre/metabolismo , Fotólise , Fotofobia , Compostos de Sulfidrila/químicaRESUMO
A new strategy is described for comparing, quantitatively, the ability of hydrogenases to tolerate exposure to O2 and anoxic oxidizing conditions. Using protein film voltammetry, the inherent sensitivities to these challenges (thermodynamic potentials and rates of reactions) have been measured for enzymes from a range of mesophilic microorganisms. In the absence of O2, all the hydrogenases undergo reversible inactivation at various potentials above that of the H+/H2 redox couple, and H2 oxidation activities are thus limited to characteristic "potential windows". Reactions with O2 vary greatly; the [FeFe]-hydrogenase from Desulfovibrio desulfuricans ATCC 7757, an anaerobe, is irreversibly damaged by O2, surviving only if exposed to O2 in the anaerobically oxidized state (which therefore affords protection). In contrast, the membrane-bound [NiFe]-hydrogenase from the aerobe, Ralstonia eutropha, reacts reversibly with O2 even during turnover and continues to catalyze H2 oxidation in the presence of O2.
Assuntos
Hidrogenase/química , Oxigênio/química , Anaerobiose , Eletroquímica , Ativação Enzimática , Hidrogênio/química , Hidrogênio/metabolismo , Hidrogenase/metabolismo , Oxirredução , Oxigênio/metabolismo , TermodinâmicaRESUMO
Production and usage of di-hydrogen, H2, in micro-organisms is catalysed by highly active, 'ancient' metalloenzymes known as hydrogenases. Based on the number and identity of metal atoms in their active sites, hydrogenases fall into three main classes, [NiFe]-, [FeFe]- and [Fe]-. All contain the unusual ligand CO (and in most cases CN- as well) making them intriguing examples of 'organometallic' cofactors. These ligands render the active sites superbly 'visible' using infrared spectroscopy, which complements the use of electron paramagnetic resonance spectroscopy in studying mechanisms and identifying intermediates. Hydrogenases are becoming a focus of attention for research into future energy technologies, not only H2 production but also H2 oxidation in fuel cells. Hydrogenases immobilized on electrodes exhibit high electrocatalytic activity, providing not only an important new technique for their investigation, but also a basis for novel fuel cells either using the enzyme itself, or inspired synthetic catalysts. Favourable comparisons have been made with platinum electrocatalysts, an advantage of enzymes being their specificity for H2 and tolerance of CO. A challenge for exploiting hydrogenases is their sensitivity to O2, but some organisms are known to produce enzymes that overcome this problem by subtle alterations of the active site and gas access channels.
Assuntos
Fontes de Energia Elétrica , Eletroquímica/instrumentação , Eletroquímica/métodos , Hidrogênio/química , Hidrogenase/química , Ferro/química , Modelos Químicos , Níquel/química , Análise Espectral/métodos , Catálise , Conformação Molecular , OxirreduçãoRESUMO
Activation of the oxidized inactive state (termed Unready or Ni(u)) of the [NiFe]-hydrogenase from Allochromatium vinosum requires removal of an unidentified oxidizing entity [O], produced by partial reduction of O(2). Dynamic electrochemical kinetic studies, subjecting enzyme molecules on an electrode to sequences of potential steps and gas injections, establish the order of events in an otherwise complex sequence of reactions that involves more than one intermediate retaining [O] or its redox equivalent; fast and reversible electron transfer precedes the rate-determining step which is followed by a reaction with H(2), or the inhibitor CO, that renders the reductive activation process irreversible.
Assuntos
Monóxido de Carbono/química , Hidrogênio/química , Hidrogenase/química , Hidrogenase/metabolismo , Monóxido de Carbono/metabolismo , Monóxido de Carbono/farmacologia , Chromatiaceae/enzimologia , Eletroquímica , Ativação Enzimática , Hidrogênio/metabolismo , Hidrogenase/antagonistas & inibidores , Cinética , Oxirredução , Oxigênio/química , Oxigênio/metabolismoRESUMO
The soluble [NiFe]-hydrogenase (SH) of the facultative lithoautotrophic proteobacterium Ralstonia eutropha H16 has up to now been described as a heterotetrameric enzyme. The purified protein consists of two functionally distinct heterodimeric moieties. The HoxHY dimer represents the hydrogenase module, and the HoxFU dimer constitutes an NADH-dehydrogenase. In the bimodular form, the SH mediates reduction of NAD(+) at the expense of H(2). We have purified a new high-molecular-weight form of the SH which contains an additional subunit. This extra subunit was identified as the product of hoxI, a member of the SH gene cluster (hoxFUYHWI). Edman degradation, in combination with protein sequencing of the SH high-molecular-weight complex, established a subunit stoichiometry of HoxFUYHI(2). Cross-linking experiments indicated that the two HoxI subunits are the closest neighbors. The stability of the hexameric SH depended on the pH and the ionic strength of the buffer. The tetrameric form of the SH can be instantaneously activated with small amounts of NADH but not with NADPH. The hexameric form, however, was also activated by adding small amounts of NADPH. This suggests that HoxI provides a binding domain for NADPH. A specific reaction site for NADPH adds to the list of similarities between the SH and mitochondrial NADH:ubiquinone oxidoreductase (Complex I).
Assuntos
Cupriavidus necator/enzimologia , Hidrogenase/química , Hidrogenase/metabolismo , NADP/metabolismo , Subunidades Proteicas/química , Sequência de Aminoácidos , Sítios de Ligação , Catálise , Sequência Conservada , Complexo I de Transporte de Elétrons/química , Ativação Enzimática , Estabilidade Enzimática , Dados de Sequência Molecular , Peso Molecular , Família Multigênica , Ligação Proteica , Homologia de Sequência de AminoácidosRESUMO
[NiFe] hydrogenases catalyze the reversible heterolytic cleavage of molecular hydrogen. Several oxidized, inactive states of these enzymes are known that are distinguishable by their very different activation properties. So far, the structural basis for this difference has not been understood because of lack of relevant crystallographic data. Here, we present the crystal structure of the ready Ni-B state of Desulfovibrio fructosovorans [NiFe] hydrogenase and show it to have a putative mu-hydroxo Ni-Fe bridging ligand at the active site. On the other hand, a new, improved refinement procedure of the X-ray diffraction data obtained for putative unready Ni-A/Ni-SU states resulted in a more elongated electron density for the bridging ligand, suggesting that it is a diatomic species. The slow activation of the Ni-A state, compared with the rapid activation of the Ni-B state, is therefore proposed to result from the different chemical nature of the ligands in the two oxidized species. Our results along with very recent electrochemical studies suggest that the diatomic ligand could be hydro-peroxide.
Assuntos
Desulfovibrio/enzimologia , Hidrogenase/química , Hidrogenase/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Modelos Moleculares , Oxirredução , Estrutura Terciária de ProteínaRESUMO
Structure and oxidation state of the Ni-Fe cofactor of the NAD-reducing soluble hydrogenase (SH) from Ralstonia eutropha were studied employing X-ray absorption spectroscopy (XAS) at the Ni K-edge, EPR, and FTIR spectroscopy. The SH comprises a nonstandard (CN)Ni-Fe(CN)(3)(CO) site; its hydrogen-cleavage reaction is resistant against inhibition by dioxygen and carbon monoxide. Simulations of the XANES and EXAFS regions of XAS spectra revealed that, in the oxidized SH, the Ni(II) is six-coordinated ((CN)O(3)S(2)); only two of the four conserved cysteines, which bind the Ni in standard Ni-Fe hydrogenases, provide thiol ligands to the Ni. Upon the exceptionally rapid reductive activation of the SH by NADH, an oxygen species is detached from the Ni; hydrogen may subsequently bind to the vacant coordination site. Prolonged reducing conditions cause the two thiols that are remote from the Ni in the native SH to become direct Ni ligands, creating a standardlike Ni(II)(CysS)(4) site, which could be further reduced to form the Ni-C (Ni(III)-H(-)) state. The Ni-C state does not seem to be involved in hydrogen cleavage. Two site-directed mutants (HoxH-I64A, HoxH-L118F) revealed structural changes at their Ni sites and were employed to further dissect the role of the extra CN ligand at the Ni. It is proposed that the predominant coordination by (CN),O ligands stabilizes the Ni(II) oxidation state throughout the catalytic cycle and is a prerequisite for the rapid activation of the SH in the presence of oxygen.