Role of a conserved glutamine residue in tuning the catalytic activity of Escherichia coli cytochrome c nitrite reductase.
The pentaheme cytochrome c nitrite reductase (NrfA) of Escherichia coli is responsible for nitrite reduction during anaerobic respiration when nitrate is scarce. The NrfA active site consists of a hexacoordinate high-spin heme with a lysine ligand on the proximal side and water/hydroxide or substrate on the distal side. There are four further highly conserved active site residues including a glutamine (Q263) positioned 8 A from the heme iron for which the side chain, unusually, coordinates a conserved, essential calcium ion. Mutation of this glutamine to the more usual calcium ligand, glutamate, results in an increase in the K m for nitrite by around 10-fold, while V max is unaltered. Protein film voltammetry showed that lower potentials were required to detect activity from NrfA Q263E when compared with native enzyme, consistent with the introduction of a negative charge into the vicinity of the active site heme. EPR and MCD spectroscopic studies revealed the high spin state of the active site to be preserved, indicating that a water/hydroxide molecule is still coordinated to the heme in the resting state of the enzyme. Comparison of the X-ray crystal structures of the as-prepared, oxidized native and mutant enzymes showed an increased bond distance between the active site heme Fe(III) iron and the distal ligand in the latter as well as changes to the structure and mobility of the active site water molecule network. These results suggest that an important function of the unusual Q263-calcium ion pair is to increase substrate affinity through its role in supporting a network of hydrogen bonded water molecules stabilizing the active site heme distal ligand.
Clarke
Thomas A
TA
Centre for Molecular and Structural Biochemistry, School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, United Kingdom.
Kemp
Gemma L
GL
Van Wonderen
Jessica H
JH
Doyle
Rose-Marie A S
RM
Cole
Jeffrey A
JA
Tovell
Nick
N
Cheesman
Myles R
MR
Butt
Julea N
JN
Richardson
David J
DJ
Hemmings
Andrew M
AM
D019943:Amino Acid Substitution; D001665:Binding Sites; D002384:Catalysis; D002942:Circular Dichroism; D003460:Crystallization; D018360:Crystallography, X-Ray; D003574:Cytochrome c Group / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000201:enzymology; D018698:Glutamic Acid / Q000737:chemistry; D005973:Glutamine / Q000737:chemistry* / Q000502:physiology; D008958:Models, Molecular
D003574:Cytochrome c Group; D005973:Glutamine; D018698:Glutamic Acid; C022302:cytochrome C-552