Reversible Protonated Resting State of the Nitrogenase Active Site
Abstract
Protonated states of the nitrogenase active site are mechanistically significant since substrate reduction is invariably accompanied by proton uptake. We report the low pH characterization by X-ray crystallography and EPR spectroscopy of the nitrogenase molybdenum iron (MoFe) proteins from two phylogenetically distinct nitrogenases (Azotobacter vinelandii, Av, and Clostridium pasteurianum, Cp) at pHs between 4.5 and 8. X-ray data at pHs of 4.5–6 reveal the repositioning of side chains along one side of the FeMo-cofactor, and the corresponding EPR data shows a new S = 3/2 spin system with spectral features similar to a state previously observed during catalytic turnover. The structural changes suggest that FeMo-cofactor belt sulfurs S3A or S5A are potential protonation sites. Notably, the observed structural and electronic low pH changes are correlated and reversible. The detailed structural rearrangements differ between the two MoFe proteins, which may reflect differences in potential protonation sites at the active site among nitrogenase species. These observations emphasize the benefits of investigating multiple nitrogenase species. Our experimental data suggest that reversible protonation of the resting state is likely occurring, and we term this state "E_0H+", following the Lowe–Thorneley naming scheme.
Additional Information
© 2017 American Chemical Society. ACS AuthorChoice - This is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes. Received: June 1, 2017; Published: July 10, 2017. This material is based upon work supported by the National Science Foundation Graduate Research Fellowship (Grant DGE-1144469 to C.N.M.), the National Institute of Health (NIH Grant GM45162 to D.C.R.), and the Howard Hughes Medical Institute (D.C.R.). We acknowledge the Gordon and Betty Moore Foundation and the Beckman Institute at Caltech for their generous support of the Molecular Observatory at Caltech. We thank the staff at Beamline 12–2, Stanford Synchrotron Radiation Lightsource (SSRL), operated for the DOE and supported by its OBER and by the NIH, NIGMS (P41GM103393), and the NCRR (P41RR001209). We thank Dr. Angelo Di Bilio for assisting with the EPR experiments, Dr. Lorenz Heidinger for providing a script for EPR simulations, Dr. Paul Oyala for assistance with the EPR simulations and analysis, and Dr. Jens Kaiser, Dr. James B. Howard, Dr. Kathryn Perez, Dr. Helen Segal, Belinda Wenke, and Renee Arias for helpful discussions. The authors declare no competing financial interest.Errata
[Page 10862. The NSF grant supporting the Caltech EPR facility was inadvertently omitted from the Acknowledgments. The correct Acknowledgments is as follows, which differs from the originally published version through the addition of a last sentence "The Caltech EPR Facility is supported by NSF-1531940."] Official citation: Correction to Reversible Protonated Resting State of the Nitrogenase Active Site Christine N. Morrison, Thomas Spatzal, and Douglas C. Rees. Journal of the American Chemical Society 2017 139 (39), 13958-13958. DOI: 10.1021/jacs.7b09849.Attached Files
Published - ja7b05695.pdf
Supplemental Material - ja7b05695_si_001.pdf
Erratum - jacs.7b09849.pdf
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Additional details
- PMCID
- PMC5553094
- Eprint ID
- 78961
- Resolver ID
- CaltechAUTHORS:20170711-141051387
- DGE-1144469
- NSF Graduate Research Fellowship
- GM45162
- NIH
- Howard Hughes Medical Institute (HHMI)
- Gordon and Betty Moore Foundation
- Caltech Beckman Institute
- Department of Energy (DOE)
- P41GM103393
- NIH
- P41RR001209
- NIH
- Created
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2017-07-11Created from EPrint's datestamp field
- Updated
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2023-10-23Created from EPrint's last_modified field