E. Claude Hatchikian

Gas access to the active site of Ni-Fe hydrogenases probed by X-ray crystallography and molecular dynamics

The 2.54 Å resolution structure of Ni-Fe hydrogenase has revealed the existence of hydrophobic channels connecting the molecular surface to the active site. A crystallographic analysis of xenon binding together with molecular dynamics simulations of xenon and H2 diffusion in the enzyme interior suggest that these channels serve as pathways for gas access to the active site.

Properties and reactivation of two different deactivated forms of Desulfovibrio gigas hydrogenase

Abstract It has previously been shown that Desulfovibrio gigas hydrogenase, as isolated, has a relatively low activity in the hydrogen-methyl viologen reductase assay, and that the activity is slowly stimulated up to 10-fold by hydrogen or strong reductants. The enzyme, before reductive activation, is also totally inactive in hydrogentritium exchange and hydrogen-dichloroindophenol (DCIP) reductase assays. The behaviour of the enzyme in various states of activation is discussed in terms of three different states: the active state, which is active in all assays, the unready state, which is totally inactive, and the ready state, which does not react with hydrogen, but which is rapidly activated by strong reductants. The conditions for the slow activation of the unready state of D. gigas hydrogenase have been investigated. The rate of activation was independent of enzyme concentration over a wide range, which rules out mechanisms involving intermolecular electron exchange. The rate was only slightly affected by pH in the range 6–9, but was strongly temperature-dependent, with activation energy 88 kJ · mol−1. The enzyme could be activated by dithiothreitol + the mediator dye, Indigo tetrasulphonate, but not by dithiothreitol alone. No effects were seen during treatments with weaker reductants, thioredoxin, iron, sulphide or nickel. These results indicate that the activation does not involve conversions of a metal centre or the cleavage of an accessible disulphide bridge. Presumably it involves an intramolecular change, possibly in the redox state or coordination of a metal centre. The active form of D. gigas hydrogenase was rapidly inactivated by oxygen, producing mostly the unready state, which could be reactivated only slowly. By contrast, anaerobic reoxidation by DCIP was able to convert the enzyme mostly to the ready state. This was identified as being inactive in hydrogen-tritium exchange and hydrogen-DCIP reductase assays but rapidly activated in the hydrogen-methyl viologen reductase assay (DCIP prevents this). It is suggested that a similar oxidation of the active enzyme may take place in the cell as a protection against oxygen.

Redox properties of the ESR‐detectable nickel in hydrogenase from Desulfovibrio gigas

Hydrogenases (EC 1.12) from numerous bacterial and algal sources are known to be iron-sulphur proteins [1]. A number of them either contain nickel [2] or require nickel for activity [3-6]. An electron-spin resonance (ESR) signal, with rhombic symmetry, has been observed in membranes of Methanobacterium bryantii [7]. The signal was tentatively assigned as low-spin Ni(III) because its g-values are all above g = 2, indicating a d 7 system, and there is no hyperfine structure [7]. This assignment was confirmed in a nickel-containing hydrogenase isolated from M. thermoautotrophicum [8] by substitution with 61Ni and observing the expected hyperfine splitting in the spectrum. The ESR spectrum therefore provides a spectroscopic probe for the properties and function of nickel in hydrogenase. Hydrogenase of Desulfovibrio gigas contains 12 atoms of iron and 12 of acid-labile sulphide in a molecule o fM r 89 500 [9]. We have now detected ~1 nickel/atom enzyme molecule. The ESR spectrum of the oxidized protein contains an intense narrow signal at g = 2.02 due to an oxidized iron-sulphur cluster [10]. We have now detected a rhombic signal which is very similar to that from nickel in M. thermoautotrophicum hydrogenase. Though broader, this signal is of significant intensity. By using redox titrations we have measured the midpoint reduction potential, E m of the ESR-detectable nickel, to be -145 mV at pH 7.2. This is higher than the I-I+/H2 couple but much lower than the E m-values normally found for Ni(III) complexes. Furthermore, the E m is pH-dependent, indicating that proton transfer takes place during reduction. The Ni(III) signal indicates no superhyperfme splittings due to interaction with nitrogenous ligands or exchangeable protons. Powe~ saturation studies indicate that the ESR-detectable nickel is distant from the ESR-detectable iron-sulphur cluster. The possibility that the nickel may be involved in the reaction cycle of the enzyme is considered.

High-resolution crystallographic analysis of Desulfovibrio fructosovorans [NiFe] hydrogenase

Abstract Two 1.8 A resolution crystal structures of an oxidised form of the [NiFe] hydrogenase of Desulfovibrio ( D .) fructosovorans are reported. The high data quality allows for a detailed analysis of the active site geometry, confirming asymmetric bridging of the Ni and Fe ion by the two cysteine sulphur atoms and one oxygen atom as previously observed in the D. gigas enzyme. The CO ligand is now clearly distinguishable from the two CN − ligands, as it refines to a significantly shorter distance to the Fe. The refined structures confirm the presence of long, mainly hydrophobic cavities that most probably provide pathways for H 2 diffusion between the molecular surface and the deeply buried active site. Amino acid sequence comparisons suggest that these cavities are significantly narrower in the so-called sensor hydrogenases, which may explain why this special class of enzymes is insensitive to O 2 .

ESR-detectable nickel and iron-sulphur centres in relation to the reversible activation of Desulfovibrio gigas hydrogenase

Abstract The electron spin resonance (ESR) spectra of hydrogenase from Desulfovibrio gigas were observed during the activation of the enzyme in the oxidized, ‘unready’, state by hydrogen. Signals from nickel(III) (Ni-A), and the [3Fe-xS] cluster were reduced within less than 5 min, and a broad ESR signal appeared at the same time. On the basis of simultaneous changes in optical absorption spectrum, it is proposed that the broad ESR signal represents one or possibly both [4Fe-4S] clusters in the reduced state. The increase of enzyme activity was much slower (at 20°C), and was accompanied by the appearance of another type of nickel signal (Ni-C), and a further small decrease and the Ni-C signal became more intense. On further reoxidation by the dye dichlorophenolindophenol at pH above 7.0 the enzyme was converted to the ‘ready’ state, which could now be reactivated much more rapidly by strong reductants. The proportion of the ready state correlated with a third type of nickel signal, Ni-B. The unready enzyme could also be slowly activated by milder reducing conditions which reduced Ni-A and the [3Fe-xS] cluster but did not induce significant amounts of the Ni-C and [4Fe-4S]1+ signals. The optical absorption changes indicate that the Ni-A is not coupled to an iron-sulphur cluster. It is proposed that the activation of the enzyme involves reduction of the nickel and possibly iron-sulphur centres, followed by a conformational change which alters the coordination state of nickel, and that the unready state contains Ni(III) in the inactive conformation, the ready state Ni(III) in the active conformation, and the active state Ni(I).

Isolation and characterization of the pyruvate-ferredoxin oxidoreductase from the sulfate-reducing bacterium Desulfovibrio africanus

We report the first purification and characterization of a pyruvate-ferredoxin oxidoreductase (POR) from a sulfate-reducing bacterium, Desulfovibrio africanus. The enzyme as isolated is highly stable in the presence of oxygen and exhibits a specific activity of 14 U/mg. D. africanus POR is a 256 kDa homodimer which contains thiamine pyrophosphate (TPP) and iron-sulfur clusters. EPR spectroscopic study of the enzyme indicates the presence of three [4Fe-4S]2+/1- centers/subunits. The midpoint potentials of the three centers are -390 mV, -515 mV and -540 mV. The catalytic mechanism of POR involves a free radical intermediate which disappears when coenzyme A is added. This behaviour is discussed in terms of an electron-transport chain from TPP to the acceptor. The enzyme activated by dithioerythritol shows an exceptionally high activity compared with other mesophile PORs and becomes very sensitive to oxygen in contrast to the enzyme before activation. The comparison of EPR spectra given by the as isolated and activated enzymes shows that neither the nature, nor the arrangement of FeS centers are affected by the activation process. D. africanus ferredoxins I and II are involved as the physiological electron carriers of the enzyme. POR was shown to be located in the cytoplasm by immunogold labelling.

[5] Nickel-iron hydrogenase

Publisher Summary This chapter describes isolation of nickel–iron [Ni–Fe] hydrogenase and its physical properties. It discusses various methods for estimation of its activity. The apparent activity of the [Ni-Fe] hydrogenases is variable, depending on the way in which they are prepared; the enzyme may be reversibly deactivated and reactivated. The changes in activity are interpreted in terms of three different forms of the enzyme, which differ in the state of the nickel center. The chapter also discusses methods for the preparation of the enzyme in these three forms. The spectroscopic properties of the three forms are summarized, with emphasis on electron paramagnetic resonance (EPR) spectra, which are particularly diagnostic of the state of the various centers in the enzyme. Sodium dodecyl sulfate (SDS) gel electrophoresis reveals that the enzyme contains two subunits of approx M r 28,000 and 62,000, respectively. The enzyme contains 1 nickel atom, 11 iron atoms, and 11 sulfide groups per molecule. Electron paramagnetic resonance analysis and Mossbauer studies indicate that the iron atoms and acid-labile sulfide present in the protein are assembled in two [4Fe-4S] 2+/1+ and one [3Fe-4S] 1+/0 cluster per molecule.

Studies of light-induced nickel EPR signals in hydrogenase: comparison of enzymes with and without selenium

Abstract Upon reduction under hydrogen-argon atmosphere, the nickel-hydrogenases generally show a characteristic rhombic EPR spectrum which is known as NiC. Illumination of this state at temperatures below 60 K has previously been shown to cause the disappearance of the Ni-C signal and the simultaneous appearance of two overlapping signals, here referred to as NiL1 and NiL2, which revert to the Ni-C state at higher temperatures. These phenomena have been compared in three nickel-containing hydrogenases, the [NiFe]-hydrogenases from Desulfovibrio gigas and Desulfovibrio fructosouorans , and the selenium-containing soluble [NiFeSe]-hydrogenase from Desulfomicrobium baculatum . Significant differences were observed between these enzymes. (1) The NiC, NiL1 and NiL2 EPR spectra were almost identical for D. gigas and D. fructosouorans hydrogenases, but the rates of photoconversion of NiC to NiL1 were different, being about 5 times slower for D. fructosouorans than for D. gigas in H 2 O. (2) The kinetic isotope effect in 2 H 2 0/H 2 0 was a factor of thirty in D. gigas , but only two in D. fructosouorans. Dm. baculatum hydrogenase showed almost no kinetic isotope effect on the NiC to NiL1 conversion, but an effect on the conversion of NiL1 to NiL2. The kinetic isotope effects indicate that the processes involve the movement of a hydrogen nucleus. (3) The NiL1 species converted to into NiL2 in the dark at a rate that was virtually temperature-independent below 30 K, indicative of a proton tunnelling process. (4) The conversion of NiL1 to NiL2 was partly reversed by light in Dm. baculatum hydrogenase, but not in the [NiFe]-hydrogenases. (5) Prolonged illumination of the three enzymes induced the appearance of a third light-induced signal, NiL3. The new signal was rhombic, with features at g = 2.41, 2.16 (the third component being unresolved) in the [NiFe]-hydrogenases and g = 2.48, 2.16, 2.03 for the [NiFeSe]-enzyme. (6) Splittings caused by by spin-spin interactions with [4Fe-4S] clusters were detected for all the illuminated signals, NiL1, NiL2 and NiL3. These were quantitatively different for the three enzymes. (7) Broadening of the NiC signals in H 2 O compared with 2 H 2 0 was observed in the g 1 and g 2 components of D. gigas and D. fructosovorans hydrogenases, but not for Dm. baculatum . This broadening effect was not seen with any of the NiL species. These comparative effects are discussed in terms of subtle differences in the structure and protein environment of the nickel site, and access to exchangeable hydrons.

Continuous monitoring of the activation and activity of [NiFe]-hydrogenases by membrane-inlet mass spectrometry

The hydrogen-deuterium (H + /D 2 ) exchange reaction catalyzed by [NiFe]-hydrogenases in the D 2 /H 2 O system has been used to study enzyme activation and activity by membrane-inlet mass spectrometry. The activation of the [NiFe]-hydrogenases from Thiocapsa roseopersicina (HynSL), Desulfovibrio fructosovorans (HynSL), Desulfomicrobium baculatum (HysSL), Rhodobacter capsulatus (HupUV), and of the bidirectional tetrameric HoxFUYH enzymes from Synechocystis PCC 6308 (Gloeocapsa alpicola) and Anabaena variabilis ATCC 29413 was determined in response to oxygen depletion and to reductant addition (molecular hydrogen, reduced methyl viologen). Natural physiological activators (NADH, NADPH) of the bidirectional [NiFe] hydrogenases could also be identified by the H + /D 2 exchange reaction. The data are discussed in the light of current models of hydrogenase catalytic mechanism.

Biochemical studies of the c-type cytochromes of the sulfate reducer Desulfovibrio africanus. Characterization of two tetraheme cytochromes c3 with different specificity

Three c-type cytochromes were isolated and characterized from the sulfate reducer Desulfovibrio africanus. A basic tetraheme cytochrome c3 of molecular mass 16 kDa was previously described and we have extended its characterization. Two other c3-type cytochromes, not previously observed, have also been characterized. These include an acidic tetraheme cytochrome c3 of molecular mass 15 kDa and an octaheme dimeric cytochrome c3 with a native size of 35 kDa. This is the first report of the presence of two distinct tetraheme cytochromes c3 in a Desulfovibrio species. The physico-chemical properties of the three cytochromes, including optical properties, iron content, cysteine and histidine content, N-terminal amino sequence and redox properties, are characteristic of cytochrome c3 family. The acidic tetraheme cytochrome c3 exhibited similar midpoint potential values for all four hemes (Em1 = -210 mV; Em2 = -240 mV; Em3 = -260 mV; Em4 = -270 mV), whereas in the basic tetraheme cytochrome c3 one heme had a much more positive potential than the others (Em1 = -90 mV; Em2 = -260 mV; Em3 = -280 mV; Em4 = -290 mV). The acidic tetraheme cytochrome c3 exhibited unique properties including amino-acid composition and poor reactivity towards hydrogenase. However, it is readily reduced by this enzyme in the presence of the basic cytochrome c3. The weak reactivity of the acidic tetraheme cytochrome c3 towards hydrogenase has been correlated with its low content of basic residues.