Elucidating the Roles of Conserved Active Site Amino Acids in the Escherichia coli Cytochrome c Nitrite Reductase

Lockwood, Colin (2013) Elucidating the Roles of Conserved Active Site Amino Acids in the Escherichia coli Cytochrome c Nitrite Reductase. Doctoral thesis, University of East Anglia.

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Abstract

The periplasmic cytochrome c nitrite reductase NrfA is a homodimeric protein containing
ten c-type cytochromes. NrfA catalyses the six electron reduction of nitrite to ammonia
which in turn facilitates anaerobic respiration. NrfA also reduces nitric oxide and
hydroxylamine to ammonium. The reduction of substrate is carried out at the distal position
of a lysine ligated heme and in an active site cavity dominated by a conserved catalytic
triad of histidine, tyrosine and arginine residues.
The role of the catalytic triad of Escherichia coli NrfA has been explored by generating NrfA
variants. Three NrfA variants were studied in which a single active site residue was
substituted: arginine to lysine (R106K), tyrosine to phenylalanine (Y216F) and histidine to
asparagine (H264N). These NrfA variants were then compared to the wild type enzyme.
The comparison of the crystal structures revealed the substituted residues in the NrfA
variants adopted similar positions to the native residues. The ability of the NrfA proteins to
reduce nitrogenous substrates was characterised by both solution assay and protein film
electrochemistry (PFE). The results revealed that R106K and Y216F NrfA maintained the
ability to reduce nitrite whereas the H264N NrfA did not. Further characterisation of H264N
NrfA using PFE identified that not only was nitrite no longer a substrate, it could instead act
as an inhibitor of hydroxylamine reduction.
A fourth variant of E. coli NrfA, in which the lysine ligand was substituted for a histidine
residue (K126H), attempted to examine the importance of lysine ligation to the active site
heme. The crystal structure of the K126H variant revealed the histidine was not ligating the
heme iron. However, spectroscopy of the K126H NrfA was unable to confirm the presence
of a tetra-coordinated heme.
The substitution of the four key residues resulted in proteins with different characteristics to
the wild type enzyme and to each other. This offered an insight into role these residues
play in the reaction mechanism of NrfA.

Item Type: Thesis (Doctoral)
Faculty \ School: Faculty of Science > School of Biological Sciences
Depositing User: Mia Reeves
Date Deposited: 12 Mar 2014 09:04
Last Modified: 12 Mar 2014 09:04
URI: https://ueaeprints.uea.ac.uk/id/eprint/48097
DOI:

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