Structure of an Actinobacterial-Type [NiFe]-Hydrogenase Reveals Insight into O2-Tolerant H2 Oxidation

A novel group of bacterial [NiFe]-hydrogenases is responsible for high-affinity H-2 uptake from the troposphere, and is therefore thought to play an important role in the global H-2 cycle. Here we present the first crystal structure at 2.85-angstrom resolution of such an actinobacterial-type hydrogenase (AH), which was isolated from the dihydrogen oxidizing bacterium, Ralstonia eutropha. The enzyme has a dimeric structure carrying two active [NiFe] sites that are interconnected by six [4Fe4S] clusters over a range of approximately 90 angstrom. Unlike most other [NiFe]-hydrogenases, the [4Fe4S] cluster proximal to the [NiFe] site is coordinated by three cysteines and one aspartate. Mutagenesis experiments revealed that this aspartate residue is related to the apparent O-2 insensitivity of the AH. Our data provide first structural insight into specialized hydrogenases that are supposed to consume atmospheric H-2 under challenging conditions, i.e. at high O-2 concentration and wide temperature and pH ranges.