Primary structure of hydrogenase I from Clostridium pasteurianum

Peptides obtained by cleavage of Clostridium pasteurianum hydrogenase I have been sequenced. The data allowed design of oligonucleotide probes which were used to clone a 2310-bp Sau3A fragment containing the hydrogenase encoding gene. The latter has been sequenced and was found to translate into a protein composed of 574 amino acids (Mr = 63,836), including 22 cysteines. C. pasteurianum hydrogenase is homologous to, but longer than, the large subunit of Desulfovibrio vulgaris (Hildenborough) [Fe] hydrogenase. It includes an additional N-terminal domain of ca. 110 amino acids which contains eight cysteine residues and which therefore could accommodate two of its postulated four [4Fe-4S] clusters. C. pasteurianum hydrogenase is most similar in length, cysteine positions, and sequence altogether to the translation product of a putative hydrogenase encoding gene from D. vulgaris (Hildenborough). Comparisons of the available [Fe] hydrogenase sequences show that these enzymes constitute a structurally rather homogeneous family. While they differ in the length of their N-termini and in the number of their [4Fe-4S] clusters, they are highly similar in their C-terminal halves, which are postulated to harbor the hydrogen-activating H cluster. Five conserved cysteine residues occurring in this domain are likely ligands of the H cluster. Possible ligation by other residues, and in particular by methionine, is discussed. The comparisons carried out here show that the H clusters most probably possess a common structural framework in all [Fe] hydrogenases. On the basis of the available data on these proteins and on the current developments in iron-sulfur chemistry, the H clusters possibly contain six to eight iron atoms.(ABSTRACT TRUNCATED AT 250 WORDS)     

Electron paramagnetic resonance studies of the low temperature photolytic behavior of oxidized hydrogenase I from Clostridium pasteurianum

The effects of CO and O2 on the EPR spectrum of oxidized Clostridium pasteurianum hydrogenase I have been investigated both before and after prolonged exposure to white light at 8 K and 30 K. Low concentrations of O2 were found to induce analogous changes in the EPR spectrum as CO, i.e. conversion of the rhombic signal with g approximately 2.10, 2.04, 2.00, a characteristic of the novel H2-activating center in oxidized Fe-hydrogenases, to an axial signal with g approximately 2.07, 2.01, 2.01. The results suggest a common binding site and mode of coordination for CO and O2 and permit rationalization of conflicting reports from different laboratories concerning the EPR properties of oxidized Fe-hydrogenases. The CO- and O2-induced axial EPR signals were found to be light-sensitive at low temperatures. Moreover, they exhibited indistinguishable and unusual photolysis behavior with the dominant photo-product being dependent on the temperature at which illumination was performed. At 8 K, photodissociation of CO or O2 occurs, resulting in an EPR signal identical with that of the oxidized enzyme in the absence of CO or O2. However, at 30 K, the dominant photoproduct is a rhombic EPR signal with g approximately 2.26, 2.12, 1.89. While the origin of this new EPR signal is uncertain, the g-value anisotropy and relaxation characteristics resemble those of a low spin Fe(III) center. These two photoproducts cannot be thermally or photolytically interconverted, but both are quantitatively reconverted to the original axial EPR signal on warming in the dark to 200 K. A tentative working hypothesis for the nature of the H2-activating center of Fe-hydrogenases is presented that is consistent with the available physiochemical data and permits rationalization of the novel photolysis behavior.     

The mechanisms of H2 activation and CO binding by hydrogenase I and hydrogenase II of Clostridium pasteurianum

Hydrogenase I (bidirectional) and hydrogenase II (uptake) of Clostridium pasteurianum have been investigated by electron paramagnetic resonance (EPR) spectroscopy, in the presence and absence of the inhibitor, CO. These hydrogenases contain both a novel type of iron-sulfur cluster (H), which is the proposed site of H2 catalysis, and ferredoxin-type [4Fe-4S] clusters (F). The results show that the H clusters of these two hydrogenases have very different properties. The H cluster of oxidized hydrogenase II (Hox-II) exhibits three distinct EPR signals, two of which are pH-dependent. Hox-II binds CO reversibly to give a single, pH-independent species with a novel, rhombic EPR spectrum. The H cluster of reduced hydrogenase II (Hred-II) does not react with CO. In contrast, the EPR spectrum of Hox-I appears homogeneous and independent of pH. Hox-I has a much lower affinity for CO than Hox-II, and binds CO irreversibly to give an axial EPR signal. Hred-I also binds CO irreversibly. The EPR spectra of Fred-I and Fred-II show little or no change after CO treatment. Prior exposure to CO does not affect the catalytic activity of the reduced or oxidized hydrogenases when assayed in the absence of CO, but both enzymes are irreversibly inactivated if CO is present during catalysis. Mechanisms for H2 activation by hydrogenase I and hydrogenase II are proposed from the determined midpoint potentials (Em, pH 8.0) of H-I and H-II (Em approximately -400 mV, -CO; approximately -360 mV, +CO), F-I (Em = -420 mV, +/- CO), and F-II (Em = -180 mV, +/- CO). These allow one to rationalize the different modes of CO binding to the two hydrogenases and suggest why hydrogenase II preferentially catalyzes H2 oxidation. The results are discussed in light of recent spectroscopic data on the structures of the two H clusters.     

An investigation of hydrogenase I and hydrogenase II from Clostridium pasteurianum by resonance Raman spectroscopy. Evidence for a [2Fe-2S] cluster in hydrogenase I

Resonance Raman spectra are reported for hydrogenase I and II from Clostridium pasteurianum. These spectra show overlapping bands with contributions from [4Fe-4S] clusters, known to be present in these enzymes, and from novel FeS centers of hitherto undefined structure. For hydrogenase I there are strong bands at 288 and 394 cm-1, which are seen in [2Fe-2S] proteins and in no other FeS species so far examined. In contrast these bands do not appear for hydrogenase II, whose resonance Raman spectrum is dominated by [4Fe-4S] cluster modes. These results provide the first structural information on the hydrogenase I FeS center involved in H2 activation and demonstrate structural differences between hydrogenase I and hydrogenase II.     

Binding of exogenously added carbon monoxide at the active site of the iron-only hydrogenase (CpI) from Clostridium pasteurianum.

A site for the binding of exogenously added carbon monoxide has been identified at the active site of the Fe-only hydrogenase (CpI) from Clostridium pasteurianum. The binding and inhibition of carbon monoxide have been exploited in biochemical and spectroscopic studies to gain mechanistic insights. In the present study, we have taken advantage of the ability to generate an irreversibly carbon monoxide bound state of CpI. The crystallization and structural characterization of CpI inhibited in the presence of carbon monoxide indicates the addition of a single molecule of carbon monoxide. The ability to generate crystals of the carbon monoxide bound state of the hydrogenase that are isomorphous to those of the native enzyme has allowed for a direct comparison of the crystallographic data and an unambiguous identification of the site of carbon monoxide binding at the active site of CpI. Carbon monoxide binds to an Fe atom of the 2Fe subcluster at the site of a terminally bound water molecule in the as crystallized native state of CpI that has been previously suggested to be a potential site of reversible hydrogen oxidation. Binding of carbon monoxide at this site results in an active site that is coordinately saturated with strong ligands (S, CO, and CN), providing a rational potential mechanism for inhibition of reversible hydrogen oxidation at the active site of CpI.     

Magnetic circular dichroism and electron paramagnetic resonance studies of hydrogenases I and II from Clostridium pasteurianum

The two iron-only hydrogenases (I and II) from Clostridium pasteurianum have been investigated by variable temperature magnetic circular dichroism (MCD) and electron paramagnetic resonance (EPR) spectroscopies. Samples were studied both reduced with dithionite under an atmosphere of H2 and after oxidation with thionine. The results are consistent with four and two [4Fe-4S]1+,2+ (F)-clusters in hydrogenases I and II, respectively. All four F-clusters are reduced and paramagnetic in reduced hydrogenase I, with up to one exhibiting an S = 3/2 ground state and the remainder having conventional S = 1/2 ground states. Both F-clusters have S = 1/2 ground states in reduced hydrogenase II; however, one appears to be only partially reduced under the conditions used for reduction. MCD studies of the oxidized enzymes show no temperature-dependent features in the visible region which can be attributed to the EPR-active S = 1/2 hydrogen-activating cluster, suggesting predominantly oxygen and nitrogen coordination for the iron atoms of this center. However, temperature-dependent MCD transitions arising from a hitherto undetected S greater than 1/2 Fe-S clusters are apparent in both oxidized hydrogenases. Detailed EPR studies of oxidized hydrogenase I revealed resonances from an S = 3/2 species, however, spin quantitation reveals this to be a trace component that is unlikely to be responsible for the observed low temperature MCD spectrum. The nature and origin of these S greater than 1/2 Fe-S clusters are discussed in light of the available spectroscopic data for these and other iron-only hydrogenases.     

1H NMR studies on the oxidized ferredoxin from Clostridium pasteurianum.

The 8Fe-8S ferredoxin from Clostridium pasteurianum was investigated by 1D and 2D 1H NMR. Spectra of a well-structured, full native preparation of the oxidized protein in 1 M NaCl at pH 8.0 are presented. Assignments of non-isotropically shifted resonances in the diamagnetic region of the spectrum, namely those of the unique aromatic residues F30 and Y2, are presented for the first time.     

An EPR and electron nuclear double resonance investigation of carbon monoxide binding to hydrogenase I (bidirectional) from Clostridium pasteurianum W5

Previous M?ssbauer and electron nuclear double resonance (ENDOR) studies of oxidized hydrogenase I (bidirectional) from Clostridium pasteurianum W5 demonstrated that this enzyme contains two diamagnetic [4Fe-4S]2+ clusters and an iron-sulfur center of unknown structure and composition that is characterized by its novel M?ssbauer and ENDOR properties. In the present study we combine ENDOR and EPR measurements to show that the novel cluster contains 3-4 iron atoms. In addition, we have used EPR and ENDOR spectroscopies to investigate the effect of binding the competitive inhibitor carbon monoxide to oxidized hydrogenase I, using 13C-labeled CO and enzyme isotopically enriched in 57Fe. Treatment of oxidized enzyme with CO causes the g-tensor of the paramagnetic center to change from rhombic to axial symmetry. The observation of a 13C signal by ENDOR spectroscopy and analysis of the EPR broadening show that a single CO covalently binds to the paramagnetic center. The 13C hyperfine coupling constant (Ac approximately equal to 21 MHz) is within the range observed for inorganic iron-carbonyl clusters. The observation of 57Fe ENDOR signals from two types of iron site ([A1c] approximately 30-34 MHz; [A2c] approximately 6 MHz) and resolved 57Fe hyperfine interactions in the EPR spectrum from two nuclei characterized by [A1c] confirm that the iron-sulfur cluster remains intact upon CO coordination, but show that CO binding greatly changes the 57Fe hyperfine coupling constants.     

Heterologous expression and properties of the gamma-subunit of the Fe-only hydrogenase from Thermotoga maritima

Thermotoga maritima is a hyperthermophilic bacterium that contains a complex, heterotrimeric (alpha(beta)gamma) Fe-only hydrogenase. Sequence analysis indicates that the gene encoding the smallest subunit (gamma), hydC, contains a predicted iron-sulfur cluster binding motif. However, characterization of the native gamma-subunit has been hampered by interference from and the inability to separate intact gamma-subunit from the other two subunits (alpha and beta). To investigate the function and properties of the isolated gamma-subunit, the gene encoding HydG was expressed in Escherichia coli. Two forms of the recombinant protein were obtained with molecular masses of 10 and 18 kDa, respectively. Both contained a single [2Fe-2S] cluster based on metal analysis, EPR and UV-visible spectroscopy. NH2-terminal sequencing revealed that the 10 kDa protein is a truncated form of the intact gamma-subunit and lacks the first 65 amino acid residues. The midpoint potential of the 18 kDa form was -356 mV at pH 7.0 and 25 degrees C, as measured by direct electrochemistry, and was pH dependent with a pK(ox) of 7.5 and a pK(red) of 7.7. The oxidized, recombinant gamma-subunit was stable at 80 degrees C under anaerobic conditions with a half-life greater than 24 h, as judged by the UV-visible spectrum of the [2Fe-2S] cluster. In the presence of air the protein was less stable and denatured with a half-life of approx. 2.5 h. The recombinant gamma-subunit was electron transfer competent and was efficiently reduced by pyruvate ferredoxin oxidoreductase from Pyrococcus furiosus, with a Km of 5microM and a Vmax of 9 U/mg. In contrast, native T. maritima hydrogenase holoenzyme and its separated alpha-subunit were much less effective electron donors for the gamma-subunit, with a V(max) of 0.01 U/mg and 0.1 U/mg, respectively.     

ADP-ribosylation of dinitrogenase reductase from Clostridium pasteurianum prevents its inhibition of nitrogenase from Azotobacter vinelandii.

Rates of protein synthesis were measured in vivo [corrected] in the lung and heart from fed rats exposed to hyperoxia (less than or equal to 95% O2) for either 6 or 24 h. Protein synthesis rates were depressed by 16-32% compared with normoxic controls in these tissues. The inhibition in both tissues was greatest after 24 h hyperoxic exposure. The decreased fractional rates of synthesis in both tissues were related to changes in ribosomal activity rather than capacity. The fall in synthesis rate per ribosome was greatest in both tissues when the exposure period was increased to 24 h. The possible mechanism(s) involved in hyperoxia-induced depression of protein synthesis are discussed.