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Electrochemical and Structural Characterization of Azotobacter vinelandii Flavodoxin II

Segal, Helen M. and Spatzal, Thomas and Hill, Michael G. and Udit, Andrew K. and Rees, Douglas C. (2017) Electrochemical and Structural Characterization of Azotobacter vinelandii Flavodoxin II. Protein Science, 26 (10). pp. 1984-1993. ISSN 0961-8368. PMCID PMC5606536. https://resolver.caltech.edu/CaltechAUTHORS:20170717-095833231

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Abstract

Azotobacter vinelandii flavodoxin II serves as a physiological reductant of nitrogenase, the enzyme system mediating biological nitrogen fixation. Wildtype A. vinelandii flavodoxin II was electrochemically and crystallographically characterized to better understand the molecular basis for this functional role. The redox properties were monitored on surfactant-modified basal plane graphite electrodes, with two distinct redox couples measured by cyclic voltammetry corresponding to reduction potentials of −483 ± 1 mV and −187 ± 9 mV (vs. NHE) in 50 mM potassium phosphate, 150 mM NaCl, pH 7.5. These redox potentials were assigned as the semiquinone/hydroquinone couple and the quinone/semiquinone couple, respectively. This study constitutes one of the first applications of surfactant-modified basal plane graphite electrodes to characterize the redox properties of a flavodoxin, thus providing a novel electrochemical method to study this class of protein. The X-ray crystal structure of the flavodoxin purified from A. vinelandii was solved at 1.17 Å resolution. With this structure, the native nitrogenase electron transfer proteins have all been structurally characterized. Docking studies indicate that a common binding site surrounding the Fe-protein [4Fe:4S] cluster mediates complex formation with the redox partners Mo-Fe protein, ferredoxin I, and flavodoxin II. This model supports a mechanistic hypothesis that electron transfer reactions between the Fe-protein and its redox partners are mutually exclusive.


Item Type:Article
Related URLs:
URLURL TypeDescription
https://doi.org/10.1002/pro.3236DOIArticle
http://onlinelibrary.wiley.com/doi/10.1002/pro.3236/abstractPublisherArticle
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5606536PubMed CentralArticle
ORCID:
AuthorORCID
Spatzal, Thomas0000-0002-9136-5915
Rees, Douglas C.0000-0003-4073-1185
Additional Information:© 2017 The Protein Society. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. Received 1 June 2017; Accepted 10 July 2017. Grant sponsor: NIH grant (D.C.R.); Grant number: GM45162; Grant sponsor: NSF Grant (A.K.U.); Grant number: 1402029; Grant sponsor: Howard Hughes Medical Institute (D.C.R.). We gratefully acknowledge discussions with Dr. James B. Howard, Dr. Jens Kaiser, Dr. Limei Zhang, Renee Arias, Christine Morrison and Belinda Wenke. We thank the Gordon and Betty Moore Foundation and the Beckman Institute for their generous support of the Molecular Observatory at Caltech, and the staff at Beamline 12–2, Stanford Synchrotron Radiation Lightsource (SSRL) for their assistance with data collection. SSRL is operated for the DOE and supported by its OBER and by the NIH, NIGMS (P41GM103393) and the NCRR (P41RR001209). Coordinates and structure factors for wildtype Azotobacter vinelandii flavodoxin II have been deposited in the Protein Data Bank of the Research Collaboratory for Structural Bioinformatics with ID 5K9B.
Funders:
Funding AgencyGrant Number
NIHGM45162
NSF1402029
Howard Hughes Medical Institute (HHMI)UNSPECIFIED
Gordon and Betty Moore FoundationUNSPECIFIED
Caltech Beckman InstituteUNSPECIFIED
Department of Energy (DOE)UNSPECIFIED
NIHP41GM103393
NIHP41RR001209
Subject Keywords:Flavodoxin; Nitrogenase; X-ray Crystallography; Electrochemistry
Issue or Number:10
PubMed Central ID:PMC5606536
Record Number:CaltechAUTHORS:20170717-095833231
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20170717-095833231
Official Citation:Segal, H. M., Spatzal, T., Hill, M. G., Udit, A. K. and Rees, D. C. (2017), Electrochemical and structural characterization of Azotobacter vinelandii flavodoxin II. Protein Science, 26: 1984–1993. doi:10.1002/pro.3236
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:79126
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:17 Jul 2017 19:24
Last Modified:14 Oct 2019 18:24

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