The hows and whys of aerobic H2 metabolism

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The bacterial [NiFe]-hydrogenases have been classified as either ‘standard’ or ‘O2-tolerant’ based on their ability to function in the presence of O2. Typically, these enzymes contain four redox-active metal centers: a Ni–Fe–CO–2CN− active site and three electron-transferring Fe–S clusters. Recent research suggests that, rather than differences at the catalytic active site, it is a novel Fe–S cluster electron transfer (ET) relay that controls how [NiFe]-hydrogenases recover from O2 attack. In light of recent structural data and mutagenic studies this article reviews the molecular mechanism of O2-tolerance in [NiFe]-hydrogenases and discusses the biosynthesis of the unique Fe–S relay.

Highlights

► A novel Fe–S cluster controls how [NiFe]-hydrogenases recover from O2 attack. ► Molecular mechanism of O2-tolerance. ► Biosynthesis of the unique Fe–S relay.

Introduction

Hydrogenases are microbial enzymes that catalyze either H2 oxidation or production. The high turnover rates and catalytic efficiencies of these enzymes are comparable to platinum, the most efficient chemical catalyst known [1•]. The most widespread hydrogenases are the [NiFe] and [FeFe] enzymes, so-named after the bi-metallic content of their active sites, and these catalyze the reversible oxidation of molecular hydrogen (H2 ↔ 2H+ + 2e−) [2]. A third class of hydrogenases, the [Fe]-only enzymes, have also been described and these catalyze heterolytic cleavage of hydrogen (H2 → H+ + H−) [3]. The active sites of all hydrogenases contain at least one Fe atom ligated by carbon monoxide and thiol (cysteine) ligands. Therefore biology uses 3d transition metals with π-accepting ligands for H2 catalysis, in contrast to the 5d transition metal Pt [4].

Most applications of H2-oxidizing and H2-producing catalysts require them to function in air. For example, an O2-stable H2-production catalyst is an essential component of an ‘artificial leaf’ – a device that uses solar energy to split water into H2 and O2 [5]. Similarly, most practical technological uses of hydrogenases would require them to remain active in the presence of O2. However, permanent O2-inactivation is an unavoidable inherent characteristic of all [FeFe]-hydrogenases because the active site [FeFe]-[4Fe–4S] ‘H-cluster’ reacts irreversibly with, and is destroyed by, O2 [6, 7]. In addition, the [Fe]-only hydrogenases are not useful in the most common technological applications of hydrogenases, which normally require a flow of electrons. These two classes of hydrogenases are therefore not discussed further in this article. The focus is on enzymes that naturally function in air. Some aerobic bacteria, such as the Knallgas bacterium Ralstonia eutropha, produce [NiFe]-hydrogenases that have evolved to operate in O2 [8, 9]. Indeed, the isolated ‘O2-tolerant’ enzymes can be used in devices like membrane-free H2/O2 fuel cells [10, 11]. This special property means that the mechanism of O2-tolerance exhibited by [NiFe]-hydrogenases has become the subject of intense research activity.

Section snippets

[NiFe]-hydrogenases

[NiFe]-hydrogenases are widespread in the microbial world and generally comprise a minimal functional unit of a large (or α-) subunit, molecular weight ∼60 kDa, which contains the [NiFe] active-site, and a small (or β-) subunit, of ∼35 kDa, which contains the Fe–S electron-transfer relay [12•, 13••, 14••]. Typically, [NiFe]-hydrogenase small subunits contain three Fe–S clusters: a distal [4Fe–4S] cluster at the surface of the protein and furthest from the [NiFe] active site; a medial [3Fe–4S]

Reaction of O2 with [NiFe]-hydrogenases

The Ni ion is redox active and this is the site of both H2 and O2 binding. A ‘standard’ [NiFe]-hydrogenase is one that is rapidly inactivated by O2, while O2-tolerant [NiFe]-hydrogenases can resist such attack. When O2 reacts at the active site of standard [NiFe]-hydrogenases two different Ni(III) inactive states can be generated: a kinetically ‘Ready’ (rapidly activating) state that is spectroscopically defined as ‘Ni-B’; and a kinetically ‘Unready’ (slow-activating) state that is

A special [4Fe–3S] cluster is responsible for O2-tolerance

There are no significant structural differences between the [NiFe] catalytic centers of O2-inactivated standard (O2-sensitive) and O2-tolerant [NiFe]-hydrogenases [13••, 14••]. Instead, it is the electron-donating capacity and tuning of the reduction potentials of the small subunit Fe–S relay that differs significantly between O2-tolerant and O2-sensitive enzymes (Figure 3) [22, 28••]. The structural fingerprint for O2-tolerance is the presence of completely conserved extra cysteine side-chains

Biosynthesis of O2-tolerant [NiFe]-hydrogenases

Now that details of the molecular basis of O2-tolerance are beginning to emerge, the challenge for biotechnologists is to harness that knowledge and so engineer O2-tolerance, or perhaps O2-resistance, into new or synthetic [NiFe]-hydrogenases. Assembly of [NiFe]-hydrogenases is, however, a complicated biochemical process.

Under anaerobic conditions the assembly of the Ni–Fe–CO–2CN− cofactor into each large subunit requires the activity of at least seven gene products each time. These include the

The HyaE/HoxO/HupG and HyaF/HoxQ/HupH families of accessory proteins

E. coli could be one of the most informative model systems for understanding the biosynthesis of O2-tolerant enzymes because the bacterium produces both O2-tolerant (Hyd-1) and standard (Hyd-2) enzymes essentially at the same time. Hyd-1 is produced from an operon of six genes, hyaABCDEF, where HyaA is the small subunit, HyaB is the large subunit and HyaC is a cytochrome that anchors the [NiFe]-hydrogenase to the membrane. There are therefore three extra genes co-expressed with Hyd-1 that are

Future perspectives

In recent years great strides have been made in understanding the mechanism by which some hydrogenases sustain H2 oxidation activity in the presence of O2. Although early studies were focused on the large subunit and its gas channels, for example single-site changes to a putative gas channel in the O2-tolerant R. eutropha MBH can make the enzyme more O2-sensitive [48], a simple gas-channel effect is not the origin of O2-tolerance in these enzymes. Instead, recent studies have proven that it is

References and recommended reading

Papers of particular interest, published within the period of review, have been highlighted as:

  • • of special interest

  • •• of outstanding interest

Acknowledgements

The BBSRC (grants H001190/1 and H003878-1) and Merton College, Oxford, are gratefully acknowledged for financial support.

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