The structure of hydrogenase-2 from Escherichia coli: implications for H2-driven proton pumping

Abstract

Under anaerobic conditions, Escherichia coli is able to metabolize molecular hydrogen via the action of several [NiFe]-hydrogenase enzymes. Hydrogenase-2, which is typically present in cells at low levels during anaerobic respiration, is a periplasmic-facing membrane-bound complex that functions as a proton pump to convert energy from hydrogen (H2) oxidation into a proton gradient; consequently, its structure is of great interest. Empirically, the complex consists of a tightly bound core catalytic module, comprising large (HybC) and small (HybO) subunits, which is attached to an Fe–S protein (HybA) and an integral membrane protein (HybB). To date, efforts to gain a more detailed picture have been thwarted by low native expression levels of Hydrogenase-2 and the labile interaction between HybOC and HybA/HybB subunits. In the present paper, we describe a new overexpression system that has facilitated the determination of high-resolution crystal structures of HybOC and, hence, a prediction of the quaternary structure of the HybOCAB complex.

Redox-dependent conformational changes of a proximal [4Fe–4S] cluster in Hyb-type [NiFe]-hydrogenase to protect the active site from O2

“…2F). 17 AvISP in Group 1e (47% identical to S77HYB) and STD-type enzyme from D. desulfuricans (DdSTD) (44% identical to S77HYB) are also O 2 -sensitive, and [4Fe-4S] P in the AOXI state were deformed. 11,18 [4Fe-4S] P of AvISP and DdSTD in AOXI have a similar structure to those of AOXI and FOXI S77HYB, respectively ( Fig.…”

Direct visible light activation of a surface cysteine-engineered [NiFe]-hydrogenase by silver nanoclusters

“…Hydrogenase-2 from E. coli is a modest H 2 producer, but one that is a valuable subject for research: notably, the ability to genetically engineer and produce this otherwise low-yielding enzyme has recently been greatly improved. 36 In conventional experiments, the recombinant enzyme evolves H 2 at a rate of approximately 6 s À1 (per active site) when driven by reduced methyl viologen at pH 6. 36 The crystal structure (PDB: 6EN9) shows Hyd-2 to be a dimer of heterodimers (ab) 2 in which the [NiFe]-active site is located in each of the two large (a) subunits and an electron relay of three FeS clusters is housed in each small (b) subunit.…”

“…36 In conventional experiments, the recombinant enzyme evolves H 2 at a rate of approximately 6 s À1 (per active site) when driven by reduced methyl viologen at pH 6. 36 The crystal structure (PDB: 6EN9) shows Hyd-2 to be a dimer of heterodimers (ab) 2 in which the [NiFe]-active site is located in each of the two large (a) subunits and an electron relay of three FeS clusters is housed in each small (b) subunit. On each a subunit there are three surfaceexposed cysteine residues that could act as tether points to link Hyd-2 to PMAA-templated AgNCs.…”

Bacterial rhomboid proteases mediate quality control of orphan membrane proteins

“…However, FtsH does not degrade inner membrane proteins with tightly folded periplasmic domains (Kihara et al , ). Of note, all rhomboid substrates we identified possess a folded periplasmic ferredoxin domain (Abaibou et al , ; Jormakka et al , ; Beaton et al , ) and so are likely to be refractory to FtsH‐mediated quality control. Rhomboids could therefore have evolved to cleave orphan substrates with periplasmic domains, complementing the activity of FtsH or indeed working in concert with other proteases as illustrated in the accompanying manuscript.…”