Membrane-Bound Hydrogenase I from the Hyperthermophilic Bacterium Aquifex aeolicus: Enzyme Activation, Redox Intermediates and Oxygen Tolerance

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)(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.

X-ray crystallographic and computational studies of the O 2 -tolerant [NiFe]-hydrogenase 1 from Escherichia coli

“…2 A and C) is caused by either a change in its overall protonation or in the position of the proton in the carboxylate moiety. These protonation changes should not affect the reduction potential of the proximal cluster, as indicated by the results of Pandelia et al (19). A protonation/deprotonation role for the equivalent Glu76 in the O 2 -sensitive hydrogenase from D. fructosovorans has been postulated (36).…”

“…A deprotonated Glu76 bound to Fe 4 (PC3 − ) did not reproduce the spectroscopic experimental data at the present level of analysis. Consequently, the proximal cluster of Aa-Hase 1, the subject of the Mössbauer and EPR spectroscopic experiments by Pandelia et al (19,20), was probably in the PC3 H and/or PC3 d − states. PC3 H and PC3 d − could therefore represent the first and second intermediates in the transfer of the Cys20 amide proton to the active site.…”

Characterization of a unique [FeS] cluster in the electron transfer chain of the oxygen tolerant [NiFe] hydrogenase from Aquifex aeolicus

“…Hase I consists of two subunits; the large subunit contains the hetero-bimetallic nickel-iron site and the small subunit the electron transfer cofactors, namely iron-sulfur clusters (6). Based on spectroelectrochemical studies, this enzyme exhibits enhanced thermostability and tolerance for inhibitors (e.g., O 2 and CO) (4,7).…”

Biosynthesis of Salmonella enterica [NiFe]-hydrogenase-5: probing the roles of system-specific accessory proteins

“…Proteins with homology to S. enterica HydH (comparable to R. 6 eutropha HoxV and Rh. leguminosarum HupK) seem to be solely required for the assembly of aerobically synthesized hydrogenases and are thought to act as scaffolds for the initial assembly of the cofactor prior to insertion into the apoenzyme.…”

“…However, a mechanistic understanding of how [NiFe]-hydrogenases can catalyse H2-oxidation in air (defined as "O2-tolerance") is well developed for periplasmically-oriented, membrane-bound [NiFe]-hydrogenases (MBH) that are produced and functional in O2, such as Salmonella enterica serovar Typhimurium [NiFe]-hydrogenase-5 (Hyd-5) [1][2][3][4][5][6][7][8]. A challenge lies in understanding how such enzymes are assembled, and the ultimate aim of such work is to provide a blueprint of the essential genes required to produce a functional [NiFe]-hydrogenase in the presence of O2.…”