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Stepwise [FeFe]-hydrogenase H-cluster assembly revealed in the structure of HydAΔEFG

Abstract

Complex enzymes containing Fe–S clusters are ubiquitous in nature, where they are involved in a number of fundamental processes including carbon dioxide fixation, nitrogen fixation and hydrogen metabolism1,2. Hydrogen metabolism is facilitated by the activity of three evolutionarily and structurally unrelated enzymes: the [NiFe]-hydrogenases, [FeFe]-hydrogenases and [Fe]-hydrogenases3,4 (Hmd). The catalytic core of the [FeFe]-hydrogenase (HydA), termed the H-cluster, exists as a [4Fe–4S] subcluster linked by a cysteine thiolate to a modified 2Fe subcluster with unique non-protein ligands5,6. The 2Fe subcluster and non-protein ligands are synthesized by the hydrogenase maturation enzymes HydE, HydF and HydG; however, the mechanism, synthesis and means of insertion of H-cluster components remain unclear7,8,9,10. Here we show the structure of HydAΔEFG (HydA expressed in a genetic background devoid of the active site H-cluster biosynthetic genes hydE, hydF and hydG) revealing the presence of a [4Fe–4S] cluster and an open pocket for the 2Fe subcluster. The structure indicates that H-cluster synthesis occurs in a stepwise manner, first with synthesis and insertion of the [4Fe–4S] subcluster by generalized host-cell machinery11,12 and then with synthesis and insertion of the 2Fe subcluster by specialized hydE-, hydF- and hydG-encoded maturation machinery7,8,9,10. Insertion of the 2Fe subcluster presumably occurs through a cationically charged channel that collapses following incorporation, as a result of conformational changes in two conserved loop regions. The structure, together with phylogenetic analysis, indicates that HydA emerged within bacteria most likely from a Nar1-like ancestor lacking the 2Fe subcluster, and that this was followed by acquisition in several unicellular eukaryotes.

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Figure 1: Ball-and-stick representation of the H-cluster in [FeFe]-hydrogenase from Clostridium pasteurianum29.
Figure 2: X-ray crystal structure of C. reinhardtii HydA ΔEFG determined to a resolution of 1.97 Å, compared with HydA from CpI.
Figure 3: Active-site comparison between C. reinhardtii HydA ΔEFG and HydA from CpI.
Figure 4: Channels for insertion into hydrogenase and nitrogenase during complex Fe–S-cluster assembly.

Accession codes

Primary accessions

Protein Data Bank

Data deposits

Coordinates and structure factors of C. reinhardtii HydAΔEFG have been deposited in the Protein Data Bank under the accession code 3LX4.

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Acknowledgements

This work was supported by a US Air Force Office of Scientific Research Multidisciplinary University Research Initiative Award (FA9550-05-01-0365, J.W.P.) and the NASA Astrobiology Institute (NAI)-funded Astrobiology Biogeocatalysis Research Center (NNA08C-N85A, J.B.B. and J.W.P.). E.S.B. was supported by a NAI postdoctoral fellowship. Portions of this research were carried out at the Stanford Synchrotron Radiation Lightsource (SSRL), a national user facility operated by Stanford University on behalf of the US Department of Energy, Office of Basic Energy Sciences. The SSRL Structural Molecular Biology programme is supported by the US Department of Energy, Office of Biological and Environmental Research, the US National Institutes of Health, National Center for Research Resources, Biomedical Technology programme, and the US National Institute of General Medical Sciences.

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Author Contributions The structural work was conducted by D.W.M. with contributions from R.S. and J.A.E. E.S.B. led the phylogenetic work with contributions from R.K.L. J.W.P. supervised the work with assistance from J.B.B. D.W.M., E.S.B. and J.W.P. led the manuscript preparation with contributions from J.B.B., R.S., R.K.L. and J.A.E.

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Correspondence to John W. Peters.

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Mulder, D., Boyd, E., Sarma, R. et al. Stepwise [FeFe]-hydrogenase H-cluster assembly revealed in the structure of HydAΔEFG. Nature 465, 248–251 (2010). https://doi.org/10.1038/nature08993

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