Electron Spin Relaxation and Biochemical Characterization
of the Hydrogenase Maturase HydF: Insights into [2Fe-2S] and [4Fe-4S]
Cluster Communication and Hydrogenase Activation
Version 2 2018-05-19, 13:18
Version 1 2017-06-12, 20:11
Posted on 2018-05-19 - 13:18
Nature utilizes [FeFe]-hydrogenase
enzymes to catalyze the interconversion
between H2 and protons and electrons. Catalysis occurs
at the H-cluster, a carbon monoxide-, cyanide-, and dithiomethylamine-coordinated
2Fe subcluster bridged via a cysteine to a [4Fe-4S] cluster. Biosynthesis
of this unique metallocofactor is accomplished by three maturase enzymes
denoted HydE, HydF, and HydG. HydE and HydG belong to the radical S-adenosylmethionine superfamily of enzymes and synthesize
the nonprotein ligands of the H-cluster. These enzymes interact with
HydF, a GTPase that acts as a scaffold or carrier protein during 2Fe
subcluster assembly. Prior characterization of HydF demonstrated the
protein exists in both dimeric and tetrameric states and coordinates
both [4Fe-4S]2+/+ and [2Fe-2S]2+/+ clusters
[Shepard, E. M., Byer, A. S., Betz, J. N., Peters, J. W., and Broderick,
J. B. (2016) Biochemistry 55, 3514–3527].
Herein, electron paramagnetic resonance (EPR) is utilized to characterize
the [2Fe-2S]+ and [4Fe-4S]+ clusters bound to
HydF. Examination of spin relaxation times using pulsed EPR in HydF
samples exhibiting both [4Fe-4S]+ and [2Fe-2S]+ cluster EPR signals supports a model in which the two cluster types
either are bound to widely separated sites on HydF or are not simultaneously
bound to a single HydF species. Gel filtration chromatographic analyses
of HydF spectroscopic samples strongly suggest the [2Fe-2S]+ and [4Fe-4S]+ clusters are coordinated to the dimeric
form of the protein. Lastly, we examined the 2Fe subcluster-loaded
form of HydF and showed the dimeric state is responsible for [FeFe]-hydrogenase
activation. Together, the results indicate a specific role for the
HydF dimer in the H-cluster biosynthesis pathway.
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Shepard, Eric M.; Byer, Amanda S.; Aggarwal, Priyanka; Betz, Jeremiah N.; Scott, Anna G.; Shisler, Krista A.; et al. (2017). Electron Spin Relaxation and Biochemical Characterization
of the Hydrogenase Maturase HydF: Insights into [2Fe-2S] and [4Fe-4S]
Cluster Communication and Hydrogenase Activation. ACS Publications. Collection. https://doi.org/10.1021/acs.biochem.7b00169
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AUTHORS (10)
ES
Eric M. Shepard
AB
Amanda S. Byer
PA
Priyanka Aggarwal
JB
Jeremiah N. Betz
AS
Anna G. Scott
KS
Krista A. Shisler
RU
Robert J. Usselman
GE
Gareth R. Eaton
SE
Sandra S. Eaton
JB
Joan B. Broderick
KEYWORDS
maturase enzymescluster types-4Sdimeric statecarrier proteindimeric formgel filtration2 Fe subcluster assemblyH-cluster biosynthesis pathway-2SH 22 Fe subcluster-loaded formtetrameric statesHydF samplesHydrogenase Maturase HydFHydF speciesBiochemical Characterizationcluster EPR signalsHydrogenase Activation Natureelectron Spin Relaxationdithiomethylamine-coordinated 2 Fe subcluster bridgedHydF dimerrelaxation timesadenosylmethionine superfamilynonprotein ligandsHydF spectroscopic samples