Abstract
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)2CO moiety. However, only four catalytic intermediates could be detected; these correspond structurally to the Ni-B, Ni-SIa, 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 H2 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.