A Chimeric NiFe Hydrogenase Heterodimer to Assess the Role of the Electron Transfer Chain in Tuning the Enzyme's Catalytic Bias and Oxygen Tolerance.

The observation that some homologous enzymes have the same active site but very different catalytic properties demonstrates the importance of long-range effects in enzyme catalysis, but these effects are often difficult to rationalize. The NiFe hydrogenases 1 and 2 (Hyd 1 and Hyd 2) from E. coli both consist of a large catalytic subunit that embeds the same dinuclear active site and a small electron-transfer subunit with a chain of three FeS clusters. Hyd 1 is mostly active in H2 oxidation and resistant to inhibitors, whereas Hyd 2 also catalyzes H2 production and is strongly inhibited by O2 and CO. Based on structural and site-directed mutagenesis data, it is currently believed that the catalytic bias and tolerance to O2 of Hyd 1 are defined by the distal and proximal FeS clusters, respectively. To test these hypotheses, we produced and characterized a hybrid enzyme made of the catalytic subunit of Hyd 1 and the electron transfer subunit of Hyd 2. We conclude that catalytic bias and sensitivity to CO are set by the catalytic subunit rather than by the electron transfer chain. We confirm the importance of the proximal cluster in making the enzyme Hyd 1 resist long-term exposure to O2, but we show that other structural determinants, in both subunits, contribute to O2 tolerance. A similar strategy based on the design of chimeric heterodimers could be used in the future to elucidate various structure-function relationships in hydrogenases and other multimeric metalloenzymes and to engineer useful hydrogenases that combine the desirable properties of distinct, homologous enzymes.

Transport limited adsorption experiments give a new lower estimate of the turnover frequency of Escherichia coli hydrogenase 1.

Protein Film Electrochemistry is a technique in which a redox enzyme is directly wired to an electrode, which substitutes for the natural redox partner. In this technique, the electrical current flowing through the electrode is proportional to the catalytic activity of the enzyme. However, in most cases, the amount of enzyme molecules contributing to the current is unknown and the absolute turnover frequency cannot be determined. Here, we observe the formation of electrocatalytically active films of E. coli hydrogenase 1 by rotating an electrode in a sub-nanomolar solution of enzyme. This process is slow, and we show that it is mass-transport limited. Measuring the rate of the immobilization allows the determination of an estimation of the turnover rate of the enzyme, which appears to be much greater than that deduced from solution assays under the same conditions.

Formate Dehydrogenases Reduce CO2 Rather than HCO3- : An Electrochemical Demonstration.

Mo/W formate dehydrogenases catalyze the reversible reduction of CO2 species to formate. It is thought that the substrate is CO2 and not a hydrated species like HCO3- , but there is still no indisputable evidence for this, in spite of the extreme importance of the nature of the substrate for mechanistic studies. We devised a simple electrochemical method to definitively demonstrate that the substrate of formate dehydrogenases is indeed CO2 .

The two CO-dehydrogenases of Thermococcus sp. AM4.

Ni-containing CO-dehydrogenases (CODHs) allow some microorganisms to couple ATP synthesis to CO oxidation, or to use either CO or CO2 as a source of carbon. The recent detailed characterizations of some of them have evidenced a great diversity in terms of catalytic properties and resistance to O2. 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 [Ni4Fe-4S] C clusters, two [4Fe-4S] B clusters and one interfacial [4Fe-4S] 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 (Km) are in the microM range for CO and in the mM range (CODH 1) or above (CODH 2) for CO2. Product inhibition is observed only for CO2 reduction, consistent with CO2 binding being much weaker than CO binding. The two enzymes are rather O2 sensitive (similarly to CODH II from Carboxydothermus hydrogenoformans), and react more slowly with O2 than any other CODH for which these data are available.

Electrochemical Characterization of a Complex FeFe Hydrogenase, the Electron-Bifurcating Hnd From Desulfovibrio fructosovorans.

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.