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Investigating Structure-Function Rel...

Hird, Krystina Chante,

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Investigating Structure-Function Relationships in Cytochrome c Nitrite Reductase: Tuning Catalysis and Redox Cooperativity /

紀錄類型:	書目-電子資源 : Monograph/item
正題名/作者:	Investigating Structure-Function Relationships in Cytochrome c Nitrite Reductase: Tuning Catalysis and Redox Cooperativity // Krystina Chante Hird.
作者:	Hird, Krystina Chante,
面頁冊數:	1 electronic resource (153 pages)
附註:	Source: Dissertations Abstracts International, Volume: 87-05, Section: B.
Contained By:	Dissertations Abstracts International87-05B.
標題:	Biochemistry. -
電子資源:	https://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=32280250
ISBN:	9798297981171

Investigating Structure-Function Relationships in Cytochrome c Nitrite Reductase: Tuning Catalysis and Redox Cooperativity /
Hird, Krystina Chante,

Investigating Structure-Function Relationships in Cytochrome c Nitrite Reductase: Tuning Catalysis and Redox Cooperativity /Krystina Chante Hird. - 1 electronic resource (153 pages)

Source: Dissertations Abstracts International, Volume: 87-05, Section: B.

Soil microbes play a crucial role in reducing nitrogen loss from agricultural systems by converting nitrate to ammonium through the dissimilatory nitrate reduction to ammonium (DNRA) pathway. Central to this important pathway in the global nitrogen cycle is the pentaheme enzyme cytochrome c nitrite reductase (NrfA). NrfA is a soluble, periplasmic enzyme that catalyzes the six-electron, eight-proton reduction of nitrite to ammonium at a single active site. This reaction is facilitated by its redox partners, quinol oxidases. In Chapter 1, I review current knowledge on the regulation, homology, and maturation of NrfA. In Chapter 2, I summarize the latest progress in elucidating the reaction mechanism of ammonia production. Building on this mechanistic understanding, I next investigated the functional roles of two key hemes within the NrfA architecture. In Chapter 3, my goal was to determine the influence of heme 1's proximal ligand on active site tuning and catalytic efficiency. We replaced the coordinating lysine with a histidine to create an all-His variant that is found natively in some DNRA organisms. As expected, this variant demonstrated greatly reduced substrate affinity and catalytic efficiency but maintained roughly half of wild-type activity levels. The catalytic changes emphasize the importance of first- and second-sphere residues on active site tuning and catalysis. In Chapter 4, I investigated the role of heme 5 which sits at the NrfA homo-dimer interface and is hypothesized to mediate electron sharing and/or electron storage. We introduced a site-directed H290M mutation, replacing the native heme 5 histidine ligand with methionine to create a functional knockout. Surprisingly, spectroscopic and electrochemical analyses revealed that this mutation significantly altered the redox potentials and reduction sequence of the hemes. These findings suggest that heme 5 may play a broader role in maintaining redox cooperativity across the hemes. Finally, in chapter 5, I characterized NrfA's redox partner and quinol oxidase, NrfH. Although still in early stages, our work on NrfH lays a foundation for future studies aimed at harnessing its electron-donating capabilities. Together, these findings advance our understanding of DNRA enzymology and may inform strategies to enhance nitrogen retention in agricultural soils.

English

ISBN: 9798297981171Subjects--Topical Terms:

518028
Biochemistry.
Subjects--Index Terms:

Soil microbes

Investigating Structure-Function Relationships in Cytochrome c Nitrite Reductase: Tuning Catalysis and Redox Cooperativity /
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520 $a Soil microbes play a crucial role in reducing nitrogen loss from agricultural systems by converting nitrate to ammonium through the dissimilatory nitrate reduction to ammonium (DNRA) pathway. Central to this important pathway in the global nitrogen cycle is the pentaheme enzyme cytochrome c nitrite reductase (NrfA). NrfA is a soluble, periplasmic enzyme that catalyzes the six-electron, eight-proton reduction of nitrite to ammonium at a single active site. This reaction is facilitated by its redox partners, quinol oxidases. In Chapter 1, I review current knowledge on the regulation, homology, and maturation of NrfA. In Chapter 2, I summarize the latest progress in elucidating the reaction mechanism of ammonia production. Building on this mechanistic understanding, I next investigated the functional roles of two key hemes within the NrfA architecture. In Chapter 3, my goal was to determine the influence of heme 1's proximal ligand on active site tuning and catalytic efficiency. We replaced the coordinating lysine with a histidine to create an all-His variant that is found natively in some DNRA organisms. As expected, this variant demonstrated greatly reduced substrate affinity and catalytic efficiency but maintained roughly half of wild-type activity levels. The catalytic changes emphasize the importance of first- and second-sphere residues on active site tuning and catalysis. In Chapter 4, I investigated the role of heme 5 which sits at the NrfA homo-dimer interface and is hypothesized to mediate electron sharing and/or electron storage. We introduced a site-directed H290M mutation, replacing the native heme 5 histidine ligand with methionine to create a functional knockout. Surprisingly, spectroscopic and electrochemical analyses revealed that this mutation significantly altered the redox potentials and reduction sequence of the hemes. These findings suggest that heme 5 may play a broader role in maintaining redox cooperativity across the hemes. Finally, in chapter 5, I characterized NrfA's redox partner and quinol oxidase, NrfH. Although still in early stages, our work on NrfH lays a foundation for future studies aimed at harnessing its electron-donating capabilities. Together, these findings advance our understanding of DNRA enzymology and may inform strategies to enhance nitrogen retention in agricultural soils.
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