The membrane-bound hydrogenase (Hase I) of the hyperthermophilic bacterium Aquifex aeolicus belongs to an intriguing class of redox enzymes that show enhanced thermostability and oxygen tolerance. Protein film electrochemistry is employed here to portray the interaction of Hase I with molecular oxygen and obtain an overall picture of the catalytic activity. Fourier transform infrared (FTIR) spectroscopy integrated with in situ electrochemistry is used to identify structural details of the [NiFe] site and the intermediate states involved in its redox chemistry. We found that the active site coordination is similar to that of standard hydrogenases, with a conserved Fe(CN)(2)CO moiety. However, only four catalytic intermediates could be detected; these correspond structurally to the Ni-B, Ni-SI(a), Ni-C, and Ni-R states of standard hydrogenases. The Ni-SI/Ni-C and Ni-C/Ni-R midpoint potentials are approximately 100 mV more positive than those observed in mesophilic hydrogenases, which may be the reason that A. aeolicus Hase I is more suitable as a catalyst for H(2) oxidation than production. Protein film electrochemistry shows that oxygen inhibits the enzyme by reacting at the active site to form a single species (Ni-B); the same inactive state is obtained under oxidizing, anaerobic conditions. The mechanism of anaerobic inactivation and reactivation in A. aeolicus Hase I is similar to that in standard hydrogenases. However, the reactivation of the former is more than 2 orders of magnitude faster despite the fact that reduction of Ni-B is not thermodynamically more favorable. A scheme for the enzymatic mechanism of A. aeolicus Hase I is presented, and the results are discussed in relation to the proposed models of oxygen tolerance.

The electrochemistry of membrane-bound [NiFe] hydrogenase I ([NiFe]-hase I) from the hyperthermophilic bacterium Aquifex aeolicus was investigated at gold and graphite electrodes. Direct and mediated H(2) oxidation were proved to be efficient in a temperature range of 25-70 degrees C, describing a potential window for H(2) oxidation similar to that of O(2)-tolerant hydrogenases. Search for enhancement of current densities and enzyme stability was achieved by the use of carbon nanotube coatings. We report high catalytic currents for H(2) oxidation up to 1 mA cm(-2), 10 times higher than at the bare electrode. Interestingly, high stability of the direct catalytic process was observed when encapsulating A. aeolicus [NiFe]-hase I into a carboxylic functionalized single walled carbon nanotube network. This suggests a peculiar interaction between the enzyme and the electrode material. The parameters that governed the orientation of the enzyme before electron transfer were thus investigated using self-assembled-monolayer gold electrodes. No control of the orientation by the charge or the hydrophobicity of the interface was demonstrated. This behavior was explained on the basis of a structural comparison between A. aeolicus [NiFe]-hase I and Desulfovibrio fructosovorans [NiFe] hydrogenase, which revealed the absence of acidic residues and an additional loop in the environment of the [4Fe-4S] distal cluster in A. aeolicus [NiFe]-hase I. Finally, the effect of inhibitors on the direct oxidation of H(2) by A. aeolicus [NiFe]-hase I encapsulated in a single walled carbon nanotube network was investigated. No inhibition by CO and tolerance toward O(2) were observed. Discussion of the reasons for such tolerance was undertaken on the basis of structural comparison with hydrogenases from aerobic bacteria.