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

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.

Name dc:type.qualificationname

phd

Level dc:type.qualificationlevel

doctoral

Grantor dc:publisher.institution

University of East Anglia

Year dc:date.issued

2013

Author dc:creator

• Lockwood, Colin

Language dc:language

en