Ni-Fe CO-dehydrogenases (CODHs) catalyze the conversion between CO and CO 2 using a chain of Fe-S clusters to mediate long-range electron transfer. One of these clusters, the D-cluster, is surface-exposed and serves to transfer electrons between CODH and external redox partners.These enzymes tend to be extremely O 2 -sensitive and are always manipulated under strictly anaerobic conditions. However, the CODH from Desulfovibrio vulgaris (Dv) appears unique: exposure to micromolar concentrations of O 2 on the minutes-timescale only reversibly inhibits the enzyme, and full activity is recovered after reduction. Here we examine whether this unusual property of Dv CODH results from the nature of its D-cluster, which is a [2Fe-2S] cluster, instead of the [4Fe-4S] cluster observed in all other characterized CODHs. To this aim we produced and characterized a Dv CODH variant where the [2Fe-2S] D-cluster is replaced with a [4Fe-4S] D-cluster through mutagenesis of the D-cluster-binding sequence motif. We determined the crystal structure of this CODH variant to 1.83-Å resolution and confirmed the incorporation of a [4Fe-4S] D-cluster. We show that upon long-term O 2 -exposure, the [4Fe-4S] D-cluster degrades whereas the [2Fe-2S] D-cluster remains intact. Crystal structures of the Dv CODH variant exposed to O 2 for increasing periods of time provide snapshots of [4Fe-4S] D-cluster degradation. We further show that the WT enzyme purified under aerobic conditions 1
Ni-containing CO-dehydrogenases (CODHs) allow some microorganisms to couple ATP synthesis to CO oxidation, or to use either CO or CO 2 as a source of carbon. The recent detailed characterizations of some of them has evidenced a great diversity in terms of catalytic properties and resistance to O 2. In an effort to increase the number of available CODHs, we have heterologously produced in Desulfovibrio fructosovorans, purified and characterized the two CooS-type CODHs (CooS1 and CooS2) from the hyperthermophilic archaeon Thermococcus sp. AM4 (Tc). We have also crystallized CooS2, which is coupled in vivo to a hydrogenase. CooS1 and CooS2 are homodimers, and harbour five metalloclusters: two NiFe 4 S 4 C clusters, two [4Fe4S] B clusters and one interfacial [4Fe4S] D cluster. We show that both are dependent on a maturase, CooC1 or CooC2, which is interchangeable. The homologous protein CooC3 does not allow Ni insertion in either CooS. The two CODHs from Tc have similar properties: they can both oxidize and produce CO. The Michaelis constants (K m) are in the microM range for CO and in the mM range (CODH 1) or above (CODH 2) for CO 2. Product inhibition is observed only for CO 2 reduction, consistent with CO 2 binding being much weaker than CO binding. The two enzymes are rather O 2 sensitive (similarly to CODH II from Carboxydothermus hydrogenoformans), and react more slowly with O 2 than any other CODH for which these data are available.
Nickel-containing enzymes are diverse in terms of function and active site structure. In many cases, the biosynthesis of the active site depends on accessory proteins which transport and insert the Ni ion. We review and discuss the literature related to the maturation of carbon monoxide dehydrogenases (CODH) which bear a nickel-containing active site consisting of a [Ni–4Fe–4S] center called the C-cluster. The maturation of this center has been much less studied than that of other nickel-containing enzymes such as urease and NiFe hydrogenase. Several proteins present in certain CODH operons, including the nickel-binding proteins CooT and CooJ, still have unclear functions. We question the conception that the maturation of all CODH depends on the accessory protein CooC described as essential for nickel insertion into the active site. The available literature reveals biological variations in CODH active site biosynthesis.
Hnd, an FeFe hydrogenase from Desulfovibrio fructosovorans, is a tetrameric enzyme that can perform flavin-based electron bifurcation. It couples the oxidation of H2 to both the exergonic reduction of NAD+ and the endergonic reduction of a ferredoxin. We previously showed that Hnd retains activity even when purified aerobically unlike other electron-bifurcating hydrogenases. In this study, we describe the purification of the enzyme under O2-free atmosphere and its biochemical and electrochemical characterization. Despite its complexity due to its multimeric composition, Hnd can catalytically and directly exchange electrons with an electrode. We characterized the catalytic and inhibition properties of this electron-bifurcating hydrogenase using protein film electrochemistry of Hnd by purifying Hnd aerobically or anaerobically, then comparing the electrochemical properties of the enzyme purified under the two conditions via protein film electrochemistry. Hydrogenases are usually inactivated under oxidizing conditions in the absence of dioxygen and can then be reactivated, to some extent, under reducing conditions. We demonstrate that the kinetics of this high potential inactivation/reactivation for Hnd show original properties: it depends on the enzyme purification conditions and varies with time, suggesting the coexistence and the interconversion of two forms of the enzyme. We also show that Hnd catalytic properties (Km for H2, diffusion and reaction at the active site of CO and O2) are comparable to those of standard hydrogenases (those which cannot catalyze electron bifurcation). These results suggest that the presence of the additional subunits, needed for electron bifurcation, changes neither the catalytic behavior at the active site, nor the gas diffusion kinetics but induces unusual rates of high potential inactivation/reactivation.
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