Oxygen Tolerance of the H2-sensing [NiFe] Hydrogenase from Ralstonia eutropha H16 Is Based on Limited Access of Oxygen to the Active Site

Thorsten Buhrke,Oliver Lenz,Norbert Krauss,Bärbel Friedrich

doi:10.1074/jbc.m503260200

Abstract

Hydrogenases, abundant proteins in the microbial world, catalyze cleavage of H2 into protons and electrons or the evolution of H2 by proton reduction. Hydrogen metabolism predominantly occurs in anoxic environments mediated by hydrogenases, which are sensitive to inhibition by oxygen. Those microorganisms, which thrive in oxic habitats, contain hydrogenases that operate in the presence of oxygen. We have selected the H2-sensing regulatory [NiFe] hydrogenase of Ralstonia eutropha H16 to investigate the molecular background of its oxygen tolerance. Evidence is presented that the shape and size of the intramolecular hydrophobic cavities leading to the [NiFe] active site of the regulatory hydrogenase are crucial for oxygen insensitivity. Expansion of the putative gas channel by site-directed mutagenesis yielded mutant derivatives that are sensitive to inhibition by oxygen, presumably because the active site has become accessible for oxygen. The mutant proteins revealed characteristics typical of standard [NiFe] hydrogenases as described for Desulfovibrio gigas and Allochromatium vinosum. The data offer a new strategy how to engineer oxygen-tolerant hydrogenases for biotechnological application.

Highlights

Hydrogenases are usually sensitive to inhibition by oxygen
Evidence is presented that the shape and size of the intramolecular hydrophobic cavities leading to the [NiFe] active site of the regulatory hydrogenase are crucial for oxygen insensitivity
Modeling of the Putative Gas Channel of the RH Based on the Structure of the D. gigas [NiFe] Hydrogenase—A crystal structure of an H2-sensing hydrogenase is not available so far

Summary

Introduction

Hydrogenases are usually sensitive to inhibition by oxygen. In particular [FeFe] hydrogenases are irreversibly destroyed by oxygen, whereas oxygen does not affect the structural integrity of [NiFe] hydrogenases but reversibly inactivates their catalytic function. On the other hand, based on spectroscopic and chemical data, the RH of R. eutropha shows a standard [NiFe] site so far as the number of CO and CNϪ ligands is concerned [14], raising the question as to what kind of mechanism may account for the oxygen tolerance of this hydrogenase. In case of [NiFe] hydrogenases, the gas channel ends close to the nickel of the active site, gated by two conserved hydrophobic amino acids, valine and leucine (Fig. 1A). These residues are highly conserved in [NiFe] hydrogenase large subunits. LacZ’, ColE1 ori RK2 ori, Tcr, Mobϩ, promoterless lacZ gene pACYC177 with a 3.3-kb PstI fragment containing hoxBC LITMUS 29 with a 2.8-kb HindIII-NcoI fragment containing PSH-hoxB-hoxC Religated pCH594 after digestion with BamHI and BglII LITMUS 28 containing a 945-bp EcoRI cut PCR product (I62V) LITMUS 28 containing a 945-bp EcoRI cut PCR product (F110L) 945-bp EcoRI fragment of pCH924 in EcoRI digested pCH861 945-bp EcoRI fragment of pCH925 in EcoRI digested pCH861 379-bp XcmI-HindIII fragment of pCH925 cloned into a 3.4-kb XcmI-HindIII fragment from pCH924 945-bp EcoRI fragment of pCH932 in EcoRI digested pCH861 Religated 5.5-kb product of inverse PCR from pCH594 228-bp SacII fragment of pCH1123 cloned into a 5.2-kb SacII fragment of pCH594 798-bp PmlI-SalI fragment of pCH928 into PmlI-SalI-treated pCH1124 798-bp PmlI-SalI fragment of pCH929 into PmlI-SalI-treated pCH1124 798-bp PmlI-SalI fragment of pCH934 into PmlI-SalI-treated pCH1124 2.68-kb HindIII-SpeI fragment from pCH1124 containing PSH-hoxBstopST-

Methods

Results

Conclusion