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Mechanism of hydrogen activation by [NiFe] hydrogenases

DOI: 10.1038/nchembio.1976 DOI Help
PMID: 26619250 PMID Help

Authors: Rhiannon M Evans (University of Oxford) , Emily J Brooke (University of Oxford) , Sara A M Wehlin (University of Oxford) , Elena Nomerotskaia (University of Oxford) , Frank Sargent (University of Dundee) , Stephen B Carr (Research Complex at Harwell) , Simon E V Phillips (Research Complex at Harwell) , Fraser A Armstrong (University of Oxford)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Nature Chemical Biology

State: Published (Approved)
Published: November 2015
Diamond Proposal Number(s): 9306

Abstract: The active site of [NiFe] hydrogenases contains a strictly conserved arginine that suspends a guanidine nitrogen atom <4.5 Å above the nickel and iron atoms. The guanidine headgroup interacts with the side chains of two conserved aspartic acid residues to complete an outer-shell canopy that has thus far proved intractable to investigation by site-directed mutagenesis. Using hydrogenase-1 from Escherichia coli, the strictly conserved residues R509 and D574 have been replaced by lysine (R509K) and asparagine (D574N) and the highly conserved D118 has been replaced by alanine (D118A) or asparagine (D118N/D574N). Each enzyme variant is stable, and their [(RS)2Niμ(SR)2Fe(CO)(CN)2] inner coordination shells are virtually unchanged. The R509K variant had >100-fold lower activity than native enzyme. Conversely, the variants D574N, D118A and D118N/D574N, in which the position of the guanidine headgroup is retained, showed 83%, 26% and 20% activity, respectively. The special kinetic requirement for R509 implicates the suspended guanidine group as the general base in H2 activation by [NiFe] hydrogenases.

Journal Keywords: Biophysics; Enzyme Mechanisms; Enzymes; X-Ray Crystallography

Subject Areas: Chemistry


Instruments: I02-Macromolecular Crystallography , I04-1-Macromolecular Crystallography (fixed wavelength) , I04-Macromolecular Crystallography