H-cluster assembly intermediates built on HydF by the radical SAM enzymes HydE and HydG

Byer, Amanda S.; Shepard, Eric M.; Ratzloff, Michael W.; Betz, Jeremiah N.; King, Paul W.; Broderick, William E.; Broderick, Joan B.

doi:10.1007/s00775-019-01709-7

H-cluster assembly intermediates built on HydF by the radical SAM enzymes HydE and HydG

Journal Article · Fri Sep 06 00:00:00 EDT 2019 · JBIC Journal of Biological Inorganic Chemistry

DOI:https://doi.org/10.1007/s00775-019-01709-7· OSTI ID:1566047

Byer, Amanda S. ^[1]; Shepard, Eric M. ^[1]; Ratzloff, Michael W. ^[2]; Betz, Jeremiah N. ^[1]; ^[2]; Broderick, William E. ^[1]; Broderick, Joan B. ^[1]

Montana State Univ., Bozeman, MT (United States)
National Renewable Energy Lab. (NREL), Golden, CO (United States)

[FeFe]-hydrogenase catalyzes the reversible reduction of protons to H₂ at a complex metallocofactor site, the H-cluster. Biosynthesis of this active-site H-cluster requires three maturation enzymes: the radical S-adenosylmethionine enzymes HydE and HydG synthesize the nonprotein ligands, while the GTPase HydF provides a scaffold for assembly of the 2Fe subcluster of the H-cluster ([2Fe]_H) prior to its transfer to hydrogenase. To delineate the assembly and delivery steps for the 2Fe precursor cluster coordinated to HydF ([2Fe]_F), we have heterologously expressed HydF in the presence of HydE alone (HydFE) or HydG alone (HydFG), and characterized the resulting purified HydF^E and HydF^G using UV-visible, EPR, and FTIR spectroscopies and biochemical assays. The iron-sulfur clusters on HydF are modified by co-expression with Hyd^E or Hyd^G, as evidenced by the changes in the visible, EPR, and FTIR spectral features. Further, biochemical assays show that HydF^E is capable of activating HydA^ΔEFG to a limited extent (~1% of WT) even though the normal source of CO and CN- ligands of [2Fe]H (HydG) was absent. Activation assays performed with HydFG, in contrast, exhibit no ability to mature HydA^ΔEFG. It appears that in the case of HydF^E, trace diatomics from the cellular environment are incorporated into a [2Fe]_F-like precursor on HydF in the absence of HydG. We conclude that the product of HydE, presumably the dithiomethylamine ligand of [2Fe]_H, is absolutely essential to the activation process, while the diatomic products of HydG can be provided from alternate sources.

View Accepted Manuscript (DOE)

Research Organization:: National Renewable Energy Laboratory (NREL), Golden, CO (United States)

Sponsoring Organization:: USDOE Office of Science (SC), Basic Energy Sciences (BES) (SC-22)

Grant/Contract Number:: AC36-08GO28308

OSTI ID:: 1566047

Report Number(s):: NREL/JA--2700-74929

Journal Information:: JBIC Journal of Biological Inorganic Chemistry, Journal Name: JBIC Journal of Biological Inorganic Chemistry Journal Issue: 6 Vol. 24; ISSN 0949-8257

Publisher:: SpringerCopyright Statement

Country of Publication:: United States

Language:: English

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H-cluster assembly intermediates built on HydF by the radical SAM enzymes HydE and HydG

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