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Title: Characterization of an M-Cluster-Substituted Nitrogenase VFe Protein

Abstract

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 enzymaticmore » 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.« less

Authors:
; ; ; ; ORCiD logo;
Publication Date:
Research Org.:
Univ. of California, Irvine, CA (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Basic Energy Sciences (BES) (SC-22)
OSTI Identifier:
1461240
Alternate Identifier(s):
OSTI ID: 1510515
Grant/Contract Number:  
SC0014470
Resource Type:
Published Article
Journal Name:
mBio (Online)
Additional Journal Information:
Journal Name: mBio (Online) Journal Volume: 9 Journal Issue: 2; Journal ID: ISSN 2150-7511
Publisher:
American Society for Microbiology
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; carbon monoxide; cofactor; hydrocarbons; molybdenum; nitrogenase; vanadium

Citation Formats

Rebelein, Johannes G., Lee, Chi Chung, Newcomb, Megan, Hu, Yilin, Ribbe, Markus W., and Lovley, ed., Derek R. Characterization of an M-Cluster-Substituted Nitrogenase VFe Protein. United States: N. p., 2018. Web. doi:10.1128/mBio.00310-18.
Rebelein, Johannes G., Lee, Chi Chung, Newcomb, Megan, Hu, Yilin, Ribbe, Markus W., & Lovley, ed., Derek R. Characterization of an M-Cluster-Substituted Nitrogenase VFe Protein. United States. doi:10.1128/mBio.00310-18.
Rebelein, Johannes G., Lee, Chi Chung, Newcomb, Megan, Hu, Yilin, Ribbe, Markus W., and Lovley, ed., Derek R. Tue . "Characterization of an M-Cluster-Substituted Nitrogenase VFe Protein". United States. doi:10.1128/mBio.00310-18.
@article{osti_1461240,
title = {Characterization of an M-Cluster-Substituted Nitrogenase VFe Protein},
author = {Rebelein, Johannes G. and Lee, Chi Chung and Newcomb, Megan and Hu, Yilin and Ribbe, Markus W. and Lovley, ed., Derek R.},
abstractNote = {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.},
doi = {10.1128/mBio.00310-18},
journal = {mBio (Online)},
number = 2,
volume = 9,
place = {United States},
year = {2018},
month = {3}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record
DOI: 10.1128/mBio.00310-18

Figures / Tables:

FIG 1 FIG 1 : Subunit and metal compositions of VnfDGKV and VnfDGKM. (A) SDS-PAGE analysis of VnfDGKV and VnfDGKM. The molecular masses (in kilodaltons) of the protein standards are shown to the left of the gel. (B) Metal contents of NifDKM, VnfDGKV, and VnfDGKM.

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    Figures/Tables have been extracted from DOE-funded journal article accepted manuscripts.