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Functional roles of the [2Fe-2S] clusters in Synechocystis PCC 6803 Hox [NiFe]-hydrogenase reactivity with ferredoxins

Journal Article · · Journal of Biological Chemistry
 [1];  [1];  [1];  [1];  [1];  [1]
  1. National Renewable Energy Laboratory (NREL), Golden, CO (United States)
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The HoxEFUYH complex of Synechocystis PCC 6803 (S. 6803) consists of a HoxEFU ferredoxin:NAD(P)H oxidoreductase subcomplex and a HoxYH [NiFe]-hydrogenase subcomplex that catalyzes reversible H2 oxidation. Prior studies have suggested that the presence of HoxE is required for reactivity with ferredoxin; however, it is unknown how HoxE is functionally integrated into the electron transfer network of the HoxEFU:ferredoxin complex. Deciphering electron transfer pathways is challenged by the rich iron-sulfur cluster content of HoxEFU, which includes a [2Fe-2S] cluster in each subunit, along with multiple [4Fe-4S] clusters and a flavin cofactor. To resolve the role of HoxE, we determined the biophysical and thermodynamic properties of each [2Fe-2S] cluster in HoxEFU using steady-state and potentiometric EPR analysis in combination with square wave voltammetry (SWV). The temperature-dependence of the EPR signal for HoxE confirmed the coordination of a single [2Fe-2S] cluster that was shown by SWV to have an Em = -424 mV (versus SHE). Strikingly, when the Em of the HoxE [2Fe-2S] cluster was analyzed in HoxEFU titrations, it was shifted by >100 mV to an Em < -525 mV (versus SHE). EPR titrations of HoxEFU gave an Em value for the [2Fe-2S] cluster of HoxF, Em = -419 mV and HoxU, Em = -349 mV. These values were used to re-analyze the diaphorase kinetics in reactions performed with ferredoxins with varying Em's. The results are formulated into a model of HoxEFU:ferredoxin reactivity and the role of HoxE in mediating electron transfer within the HoxEFU:ferredoxin complex.

Research Organization:
National Renewable Energy Laboratory (NREL), Golden, CO (United States)
Sponsoring Organization:
USDOE Office of Science (SC), Basic Energy Sciences (BES). Chemical Sciences, Geosciences & Biosciences Division (CSGB); USDOE Office of Science (SC), Office of Workforce Development for Teachers & Scientists (WDTS)
Grant/Contract Number:
AC36-08GO28308
OSTI ID:
2561249
Report Number(s):
NREL/JA--2700-90401; MainId:92179; UUID:73a0e0b6-ead4-452c-b571-469136f5ca54; MainAdminId:74091
Journal Information:
Journal of Biological Chemistry, Journal Name: Journal of Biological Chemistry Journal Issue: 12 Vol. 300; ISSN 0021-9258
Publisher:
American Society for Biochemistry and Molecular BiologyCopyright Statement
Country of Publication:
United States
Language:
English

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Related Subjects

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY
59 BASIC BIOLOGICAL SCIENCES
BASIC BIOLOGICAL SCIENCES,INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY
EPR
Synechocystis
[NiFe]-hydrogenase
diaphorase
electron transfer
ferredoxin
iron-sulfur cluster
redox potential
square wave voltammetry