Hydrogenases are essential for H(2) cycling in microbial metabolism and serve as valuable blueprints for H(2)-based biotechnological applications. However, most hydrogenases are extremely oxygen sensitive and prone to inactivation by even traces of O(2). The O(2)-tolerant membrane-bound [NiFe]-hydrogenase of Ralstonia eutropha H16 is one of the few examples that can perform H(2) uptake in the presence of ambient O(2). Here we show that O(2) tolerance is crucially related to a modification of the internal electron-transfer chain. The iron-sulfur cluster proximal to the active site is surrounded by six instead of four conserved coordinating cysteines. Removal of the two additional cysteines alters the electronic structure of the proximal iron-sulfur cluster and renders the catalytic activity sensitive to O(2) as shown by physiological, biochemical, spectroscopic and electrochemical studies. The data indicate that the mechanism of O(2) tolerance relies on the reductive removal of oxygenic species guided by the unique architecture of the electron relay rather than a restricted access of O(2) to the active site.

Use of hydrogen in fuel cells requires catalysts that are tolerant to oxygen and are able to function in the presence of poisons such as carbon monoxide. Hydrogen-cycling catalysts are widespread in the bacterial world in the form of hydrogenases, enzymes with unusual active sites composed of iron, or nickel and iron, that are buried within the protein. We have established that the membrane-bound hydrogenase from the ␤-proteobacterium Ralstonia eutropha H16, when adsorbed at a graphite electrode, exhibits rapid electrocatalytic oxidation of hydrogen that is completely unaffected by carbon monoxide [at 0.9 bar (1 bar ‫؍‬ 100 kPa), a 9-fold excess] and is inhibited only partially by oxygen. The practical significance of this discovery is illustrated with a simple fuel cell device, thus demonstrating the feasibility of future hydrogen-cycle technologies based on biological or biologically inspired electrocatalysts having high selectivity for hydrogen.biohydrogen ͉ electron transfer ͉ energy ͉ fuel cell ͉ hydrogenase H ydrogenases prevail throughout the microbial world and are essential to hydrogen metabolism (1). X-ray crystallography in conjunction with Fourier transform infrared spectroscopy has revealed that the active site structure of the [NiFe] hydrogenases from Desulfovibrio gigas (Dg) (2) and D. vulgaris Miyazaki F (3, 4) is a bimetallic site having Ni and Fe centers linked by bridging cysteinyl S, with the Fe additionally coordinated by one CO and two CN Ϫ ligands. Crystallographic experiments on the enzyme from D. fructosovorans involving infusion of Xe have revealed the likely positions of ''gas channels'' for transport of small molecules such as H 2 , O 2 , and CO to and from the active site (5).The vast majority of hydrogenase-containing microorganisms, including the Desulfovibrio species, live under anaerobic or semianaerobic conditions, and like their hosts, the hydrogenases are usually highly sensitive to O 2 (1). However, some bacteria are able to gain energy from H 2 oxidation under aerobic conditions. A well studied example is Ralstonia eutropha (Re, formerly Alcaligenes eutrophus) strain H16 a ␤-proteobacterium that hosts three physiologically distinct [NiFe] hydrogenases (6-8). One of these enzymes, the membrane-bound hydrogenase (MBH), is coupled via a b-type cytochrome to the respiratory chain. Sequence similarity shows that this enzyme belongs to the family of [NiFe] hydrogenases having a large subunit containing the NiOFe catalytic center and a small electron-transferring subunit accommodating three iron-sulfur clusters (9). The MBH enables Re to grow on H 2 as the sole energy source even under ambient levels of O 2 . The exceptional tolerance of Re MBH to O 2 (10) inspired us to assess its electrocatalytic activity under extremely demanding conditions, including the presence of CO, which is the classic inhibitor of hydrogen-cycling catalysts (11). This inhibition is rationalized on the basis that activation of H 2 by transition metals requires it to form a bond that involves back donation ...

Hydrogenases are metalloenzymes that catalyze the reversible cleavage of H 2 into protons and electrons and play a pivotal role in the energy metabolism of many microorganisms (1). They are grouped into three phylogenetically distinct classes as follows: the di-iron [FeFe], nickel-iron [NiFe], and iron-sulfur cluster-free [Fe] hydrogenases (2-6). The basic module of [NiFe] hydrogenases consists of two subunits, a large subunit that contains the [NiFe] active site and a small subunit that accommodates one to three electron-transferring iron-sulfur