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.

Catalytic long-range proton transfer in [NiFe]-hydrogenases has long been associated with a highly conserved glutamate (E) situated within 4 Å of the active site. Substituting for glutamine (Q) in the O-tolerant [NiFe]-hydrogenase-1 from Escherichia coli produces a variant (E28Q) with unique properties that have been investigated using protein film electrochemistry, protein film infrared electrochemistry, and X-ray crystallography. At pH 7 and moderate potential, E28Q displays approximately 1% of the activity of the native enzyme, high enough to allow detailed infrared measurements under steady-state conditions. Atomic-level crystal structures reveal partial displacement of the amide side chain by a hydroxide ion, the occupancy of which increases with pH or under oxidizing conditions supporting formation of the superoxidized state of the unusual proximal [4Fe-3S] cluster located nearby. Under these special conditions, the essential exit pathway for at least one of the H ions produced by H oxidation, and assumed to be blocked in the E28Q variant, is partially repaired. During steady-state H oxidation at neutral pH (i.e., when the barrier to H exit via Q28 is almost totally closed), the catalytic cycle is dominated by the reduced states "Ni-R" and "Ni-C", even under highly oxidizing conditions. Hence, E28 is not involved in the initial activation/deprotonation of H, but facilitates H exit later in the catalytic cycle to regenerate the initial oxidized active state, assumed to be Ni-SI. Accordingly, the oxidized inactive resting state, "Ni-B", is not produced by E28Q in the presence of H at high potential because Ni-SI (the precursor for Ni-B) cannot accumulate. The results have important implications for understanding the catalytic mechanism of [NiFe]-hydrogenases and the control of long-range proton-coupled electron transfer in hydrogenases and other enzymes.

Engineering a cysteine close to the distal [4Fe–4S] cluster of a [NiFe]-hydrogenase creates a specific target for Ag nanoclusters, the resulting ‘hard-wired’ enzyme catalyzing rapid hydrogen evolution by visible light.

Although multiprotein membrane complexes play crucial roles in bacterial physiology and virulence, the mechanisms governing their quality control remain incompletely understood. In particular, it is not known how unincorporated, orphan components of protein complexes are recognised and eliminated from membranes. Rhomboids, the most widespread and largest superfamily of intramembrane proteases, are known to play key roles in eukaryotes. In contrast, the function of prokaryotic rhomboids has remained enigmatic. Here, we show that the Shigella sonnei rhomboid proteases GlpG and the newly identified Rhom7 are involved in membrane protein quality control by specifically targeting components of respiratory complexes, with the metastable transmembrane domains (TMDs) of rhomboid substrates protected when they are incorporated into a functional complex. Initial cleavage by GlpG or Rhom7 allows subsequent degradation of the orphan substrate. Given the occurrence of this strategy in an evolutionary ancient organism and the presence of rhomboids in all domains of life, it is likely that this form of quality control also mediates critical events in eukaryotes and protects cells from the damaging effects of orphan proteins.

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Changing the conserved active-site arginine of [NiFe]-hydrogenases into a lysine greatly lowers the rates of catalytic H2 activation in each direction and results in the extremely tight binding of a diatomic ligand.

The photosynthetic bacterium Rhodobacter capsulatus normally photoproduces H as a by-product of its nitrogenase-catalyzed nitrogen-fixing activity. Such H production, however, is expensive from a metabolic perspective, requiring nearly four times as many photons as the equivalent algal hydrogenase-based system (Ghirardi et al., 2009 Photobiological hydrogen-producing systems. Chem Soc Rev 38(1):52-61). Here, we report the insertion of a Clostridium acetobutylicum [FeFe]-hydrogenase and its three attendant hydrogenase assembly proteins into an R. capsulatus strain lacking its native uptake hydrogenase. Further, this strain is modified to fluoresce upon sensing H . The resulting strain photoproduces H and self-reports its own H production through fluorescence. This model system represents a unique method of developing hydrogenase-based H production in R. capsulatus, may serve as a powerful system for in vivo directed evolution of hydrogenases and hydrogenase-associated genes, and provides a means of screening for increased metabolic production of H . Biotechnol. Bioeng. 2017;114: 291-297. © 2016 Wiley Periodicals, Inc.