Oxygen-tolerant H2 Oxidation by Membrane-bound [NiFe] Hydrogenases of Ralstonia Species: COPING WITH LOW LEVEL H2 IN AIR

Marcus Ludwig,James A Cracknell,Kylie A Vincent,Fraser A Armstrong,Oliver Lenz

doi:10.1074/jbc.m803676200

Marcus Ludwig, James A Cracknell + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m803676200

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Abstract

Knallgas bacteria such as certain Ralstonia spp. are able to obtain metabolic energy by oxidizing trace levels of H2 using O2 as the terminal electron acceptor. The [NiFe] hydrogenases produced by these organisms are unusual in their ability to oxidize H2 in the presence of O2, which is a potent inactivator of most hydrogenases through attack at the active site. To probe the origin of this unusual O2 tolerance, we conducted a study on the membrane-bound hydrogenase from Ralstonia eutropha H16 and that of the closely related organism Ralstonia metallidurans CH34, which was purified using a new heterologous overproduction system. Direct electrochemical methods were used to determine apparent inhibition constants for O2 inhibition of H2 oxidation (K I(app)O2) for each enzyme. These values were at least 2 orders of magnitude higher than those of "standard" [NiFe] hydrogenases. Amino acids close to the active site were exchanged in the membrane-bound hydrogenase of R. eutropha H16 for those from standard hydrogenases to probe the role of individual residues in conferring O2 sensitivity. Michaelis constants for H2 (K M H2) were determined, and for some mutants these were increased more than 20-fold relative to the wild type. Mutations resulting in membrane-bound hydrogenase enzymes with increased K M H2 or decreased K I(app)O2 values were associated with impaired lithoautotrophic growth in the presence of high O2 concentrations.

Highlights

Hydrogenases are active catalysts both in H2 oxidation and proton reduction [1]
New Protocol for Purification of MBH Proteins from Re H16 and Rm CH34—In Re H16 the genes coding for the MBH subunits, specific accessory proteins, a set of Hyp proteins, and the H2-sensing apparatus are clustered in a single operon (Fig. 2), which maps on megaplasmid pHG1 [29, 39, 40]
The gene organization of the Rm CH34 MBH gene cluster, which is located on chromosome 1 (DOE Joint Genome Institute), resembles that of Re H16 with few exceptions (Fig. 2)

Summary

Introduction

Hydrogenases are active catalysts both in H2 oxidation and proton reduction [1]. Three phylogenetically distinct classes of hydrogenases are found in nature as follows: [FeFe], [NiFe], and [Fe] hydrogenases [2, 3]. Dioxygen is a ␲-acceptor ligand like H2 and CO and is expected to enter the active site It subsequently behaves as an oxidizing agent, leading to inactive “resting” states. A practical demonstration of this O2 tolerance was provided by the construction of an H2 fuel cell employing Rm CH34 MBH as the anode catalyst and laccase (a multicopper oxidase catalyzing the clean four-electron reduction of O2 to water) at the cathode. With this device it was possible to produce sufficient electricity from a quiescent atmosphere of just 3% H2 in air to power a wristwatch for over 24 h [19]

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