Characterization of an M-Cluster-Substituted Nitrogenase VFe Protein

ABSTRACT The Mo- and V-nitrogenases are two homologous members of the nitrogenase family that are distinguished mainly by the presence of different heterometals (Mo or V) at their respective cofactor sites (M- or V-cluster). However, the V-nitrogenase is ~600-fold more active than its Mo counterpart...

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Autores principales:	Johannes G. Rebelein, Chi Chung Lee, Megan Newcomb, Yilin Hu, Markus W. Ribbe
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2018
Materias:	carbon monoxide cofactor hydrocarbons molybdenum nitrogenase vanadium Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/6a83175dce164f8ca47194c547c442a6
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spelling	oai:doaj.org-article:6a83175dce164f8ca47194c547c442a62021-11-15T15:53:26ZCharacterization of an M-Cluster-Substituted Nitrogenase VFe Protein10.1128/mBio.00310-182150-7511https://doaj.org/article/6a83175dce164f8ca47194c547c442a62018-05-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.00310-18https://doaj.org/toc/2150-7511ABSTRACT The Mo- and V-nitrogenases are two homologous members of the nitrogenase family that are distinguished mainly by the presence of different heterometals (Mo or V) at their respective cofactor sites (M- or V-cluster). However, the V-nitrogenase is ~600-fold more active than its Mo counterpart in reducing CO to hydrocarbons at ambient conditions. Here, we expressed an M-cluster-containing, hybrid V-nitrogenase in Azotobacter vinelandii and compared it to its native, V-cluster-containing counterpart in order to assess the impact of protein scaffold and cofactor species on the differential reactivities of Mo- and V-nitrogenases toward CO. Housed in the VFe protein component of V-nitrogenase, the M-cluster displayed electron paramagnetic resonance (EPR) features similar to those of the V-cluster and demonstrated an ~100-fold increase in hydrocarbon formation activity from CO reduction, suggesting a significant impact of protein environment on the overall CO-reducing activity of nitrogenase. On the other hand, the M-cluster was still ~6-fold less active than the V-cluster in the same protein scaffold, and it retained its inability to form detectable amounts of methane from CO reduction, illustrating a fine-tuning effect of the cofactor properties on this nitrogenase-catalyzed reaction. Together, these results provided important insights into the two major determinants for the enzymatic activity of CO reduction while establishing a useful framework for further elucidation of the essential catalytic elements for the CO reactivity of nitrogenase. IMPORTANCE This is the first report on the in vivo generation and in vitro characterization of an M-cluster-containing V-nitrogenase hybrid. The “normalization” of the protein scaffold to that of the V-nitrogenase permits a direct comparison between the cofactor species of the Mo- and V-nitrogenases (M- and V-clusters) in CO reduction, whereas the discrepancy between the protein scaffolds of the Mo- and V-nitrogenases (MoFe and VFe proteins) housing the same cofactor (M-cluster) allows for an effective assessment of the impact of the protein environment on the CO reactivity of nitrogenase. The results of this study provide a first look into the “weighted” contributions of protein environment and cofactor properties to the overall activity of CO reduction; more importantly, they establish a useful platform for further investigation of the structural elements attributing to the CO-reducing activity of nitrogenase.Johannes G. RebeleinChi Chung LeeMegan NewcombYilin HuMarkus W. RibbeAmerican Society for Microbiologyarticlecarbon monoxidecofactorhydrocarbonsmolybdenumnitrogenasevanadiumMicrobiologyQR1-502ENmBio, Vol 9, Iss 2 (2018)
institution	DOAJ
collection	DOAJ
language	EN
topic	carbon monoxide cofactor hydrocarbons molybdenum nitrogenase vanadium Microbiology QR1-502
spellingShingle	carbon monoxide cofactor hydrocarbons molybdenum nitrogenase vanadium Microbiology QR1-502 Johannes G. Rebelein Chi Chung Lee Megan Newcomb Yilin Hu Markus W. Ribbe Characterization of an M-Cluster-Substituted Nitrogenase VFe Protein
description	ABSTRACT The Mo- and V-nitrogenases are two homologous members of the nitrogenase family that are distinguished mainly by the presence of different heterometals (Mo or V) at their respective cofactor sites (M- or V-cluster). However, the V-nitrogenase is ~600-fold more active than its Mo counterpart in reducing CO to hydrocarbons at ambient conditions. Here, we expressed an M-cluster-containing, hybrid V-nitrogenase in Azotobacter vinelandii and compared it to its native, V-cluster-containing counterpart in order to assess the impact of protein scaffold and cofactor species on the differential reactivities of Mo- and V-nitrogenases toward CO. Housed in the VFe protein component of V-nitrogenase, the M-cluster displayed electron paramagnetic resonance (EPR) features similar to those of the V-cluster and demonstrated an ~100-fold increase in hydrocarbon formation activity from CO reduction, suggesting a significant impact of protein environment on the overall CO-reducing activity of nitrogenase. On the other hand, the M-cluster was still ~6-fold less active than the V-cluster in the same protein scaffold, and it retained its inability to form detectable amounts of methane from CO reduction, illustrating a fine-tuning effect of the cofactor properties on this nitrogenase-catalyzed reaction. Together, these results provided important insights into the two major determinants for the enzymatic activity of CO reduction while establishing a useful framework for further elucidation of the essential catalytic elements for the CO reactivity of nitrogenase. IMPORTANCE This is the first report on the in vivo generation and in vitro characterization of an M-cluster-containing V-nitrogenase hybrid. The “normalization” of the protein scaffold to that of the V-nitrogenase permits a direct comparison between the cofactor species of the Mo- and V-nitrogenases (M- and V-clusters) in CO reduction, whereas the discrepancy between the protein scaffolds of the Mo- and V-nitrogenases (MoFe and VFe proteins) housing the same cofactor (M-cluster) allows for an effective assessment of the impact of the protein environment on the CO reactivity of nitrogenase. The results of this study provide a first look into the “weighted” contributions of protein environment and cofactor properties to the overall activity of CO reduction; more importantly, they establish a useful platform for further investigation of the structural elements attributing to the CO-reducing activity of nitrogenase.
format	article
author	Johannes G. Rebelein Chi Chung Lee Megan Newcomb Yilin Hu Markus W. Ribbe
author_facet	Johannes G. Rebelein Chi Chung Lee Megan Newcomb Yilin Hu Markus W. Ribbe
author_sort	Johannes G. Rebelein
title	Characterization of an M-Cluster-Substituted Nitrogenase VFe Protein
title_short	Characterization of an M-Cluster-Substituted Nitrogenase VFe Protein
title_full	Characterization of an M-Cluster-Substituted Nitrogenase VFe Protein
title_fullStr	Characterization of an M-Cluster-Substituted Nitrogenase VFe Protein
title_full_unstemmed	Characterization of an M-Cluster-Substituted Nitrogenase VFe Protein
title_sort	characterization of an m-cluster-substituted nitrogenase vfe protein
publisher	American Society for Microbiology
publishDate	2018
url	https://doaj.org/article/6a83175dce164f8ca47194c547c442a6
work_keys_str_mv	AT johannesgrebelein characterizationofanmclustersubstitutednitrogenasevfeprotein AT chichunglee characterizationofanmclustersubstitutednitrogenasevfeprotein AT megannewcomb characterizationofanmclustersubstitutednitrogenasevfeprotein AT yilinhu characterizationofanmclustersubstitutednitrogenasevfeprotein AT markuswribbe characterizationofanmclustersubstitutednitrogenasevfeprotein
_version_	1718427280464674816

Characterization of an M-Cluster-Substituted Nitrogenase VFe Protein

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