Affiliation:
1. Microbial Metalloenzymes Research Group, Max Planck Institute for Terrestrial Microbiology, Marburg, Germany
2. Core Facility for Mass Spectrometry & Proteomics, Max Planck Institute for Terrestrial Microbiology, Marburg, Germany
3. Evolutionary Biochemistry Research Group, Max Planck Institute for Terrestrial Microbiology, Marburg, Germany
4. Center for Synthetic Microbiology (SYNMIKRO), Philipps University Marburg, Marburg, Germany
Abstract
ABSTRACT
Nitrogenases are the only enzymes able to fix gaseous nitrogen into bioavailable ammonia and hence are essential for sustaining life. Catalysis by nitrogenases requires both a large amount of ATP and electrons donated by strongly reducing ferredoxins or flavodoxins. Our knowledge about the mechanisms of electron transfer to nitrogenase enzymes is limited: The electron transport to the iron (Fe)-nitrogenase has hardly been investigated. Here, we characterized the electron transfer pathway to the Fe-nitrogenase in
Rhodobacter capsulatus via
proteome analyses, genetic deletions, complementation studies, and phylogenetics. Proteome analyses revealed an upregulation of four ferredoxins under nitrogen-fixing conditions reliant on the Fe-nitrogenase in a molybdenum nitrogenase knockout strain, compared to non-nitrogen-fixing conditions. Based on these findings,
R. capsulatus
strains with deletions of ferredoxin (
fdx
) and flavodoxin (
fld, nifF
) genes were constructed to investigate their roles in nitrogen fixation by the Fe-nitrogenase.
R. capsulatus
deletion strains were characterized by monitoring diazotrophic growth and Fe-nitrogenase activity
in vivo
. Only deletions of
fdxC
or
fdxN
resulted in slower growth and reduced Fe-nitrogenase activity, whereas the double deletion of both
fdxC
and
fdxN
abolished diazotrophic growth. Differences in the proteomes of ∆
fdxC
and ∆
fdxN
strains, in conjunction with differing plasmid complementation behaviors of
fdxC
and
fdxN,
indicate that the two Fds likely possess different roles and functions. These findings will guide future engineering of the electron transport systems to nitrogenase enzymes, with the aim of increased electron flux and product formation.
IMPORTANCE
Nitrogenases are essential for biological nitrogen fixation, converting atmospheric nitrogen gas to bioavailable ammonia. The production of ammonia by diazotrophic organisms, harboring nitrogenases, is essential for sustaining plant growth. Hence, there is a large scientific interest in understanding the cellular mechanisms for nitrogen fixation
via
nitrogenases. Nitrogenases rely on highly reduced electrons to power catalysis, although we lack knowledge as to which proteins shuttle the electrons to nitrogenases within cells. Here, we characterized the electron transport to the iron (Fe)-nitrogenase in the model diazotroph
Rhodobacter capsulatus
, showing that two distinct ferredoxins are very important for nitrogen fixation despite having different redox centers. In addition, our research expands upon the debate on whether ferredoxins have functional redundancy or perform distinct roles within cells. Here, we observe that both essential ferredoxins likely have distinct roles based on differential proteome shifts of deletion strains and different complementation behaviors.
Funder
Deutsche Forschungsgemeinschaft
Max Planck Society
Publisher
American Society for Microbiology
Cited by
2 articles.
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