Insights into the electron transport proteins essential for nitrogen fixation
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Philipps-Universität Marburg
Abstract
Nitrogen fixation, the process of converting inert gaseous nitrogen (N2) into bioavailable ammonia
(NH3) is essential for all life on earth. Nitrogen is an indispensable element for biology, being an
integral part of amino acids, the building blocks of proteins, and nitrogenous nucleotide bases,
the key components of genetic material. Despite this, nitrogen fixation is a unique feature of only
some microorgansisms called ‘diazotrophs’. Such diazotrophic organisms are vital for the
maintenance of earth’s ecosystems, being the key first step in the global nitrogen cycle.
The enzymes that catalyse this biological nitrogen fixation are the nitrogenases, which are
metalloenzymes harbouring several intricate inorganic cofactors. There are three isoforms of
nitrogenase enyzmes known: the canonical molybdenum (Mo)-nitrogenase and two alternative
nitrogenases, the vanadium (V)-nitrogenase and the iron only (Fe)-nitrogenase. The alternative
nitrogenase isoforms are understudied relative to the Mo-nitrogenase due to their later discovery
and lower catalytic efficiencies for nitrogen fixation. Despite this, the alternative nitrogenases have
important roles under Mo-deplete conditions and show interesting side reactivities with other nonnitrogenous
gases such as carbon dioxide.
Due to their unique activities, nitrogenases have been highly sought-after research targets for
many decades. However, the complex nature of nitrogenases, in terms of their protein structures,
metallocluster structures and their intricate enzymatic maturation, means many unanswered
research questions remain.
One key area of nitrogenase research where knowledge is limited is in the mechanisms of electron
delivery to nitrogenases in vivo. The delivery of high-energy electrons to nitrogenases is essential
for nitrogen fixation, with nitrogenase catalysis requiring both low potential electrons and chemical
energy created by the hydrolysis of adenosine triphosphate (ATP). The low potential electrons
are shuttled to nitrogenases by soluble electron carriers called ferredoxins and flavodoxins.
Despite the importance of these transport mechanisms, only electron transport by ferredoxins and
flavodoxins to the Mo-nitrogenase has been thoroughly characterised. The features and
mechanisms of the electron transport systems to the alternative nitrogenases have not been
thoroughly explored. Prior to this work, it had not been established which soluble electron carriers
shuttle electrons to the Fe-nitrogenase in any organism.
This thesis reports the systematic characterisation of the electron transport systems for nitrogen
fixation by the Fe-nitrogenase, within the photosynthetic diazotrophic bacterium Rhodobacter
capsulatus. Chapter two details the use of microbiological techniques, primarily genetic deletion
Summary
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construction and whole cell proteomics, to identify two distinct essential ferredoxins, FdC and
FdN, for Fe-nitrogenase mediated nitrogen fixation. The two ferredoxins are hypothesised to fulfil
differing roles within the cell, supported by a combination of phylogenetic analysis, plasmid
complementation studies and proteomics experiments. Chapter three builds upon this
hypothesis by uncovering key differences between the structures and electronic properties of FdC
and FdN through biochemical characterisations in vitro. Finally, chapter four describes efforts
towards developing proteomics-based methods for identifying ferredoxin interactions within R.
capsulatus, to map the electron transport pathways occurring during nitrogen fixation.
Overall, this work has created a foundation of knowledge for the future study of ferredoxins
essential for nitrogen fixation. We reveal novel details about the biophysical features of nitrogen
fixation-related ferredoxins, in terms of structure and electronic properties, providing key insights
into how these proteins drive nitrogen fixation. Finally, this work identifies two interesting protein
targets for engineering the electron transport systems to the Fe-nitrogenase in vivo, with aims to
increase electron flux and thus product formation.
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