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No intermediates become liberated in the course of this multi-electron, multi-
proton reduction process, such as nitric oxide (NO) or hydroxylamine (H
2
NOH).
This requires a remarkable flexibility of the active site combined with a finely tuned
proton and electron delivery system. However, NrfA will convert NO and H
2
NOH
to ammonia, and it will react with N
2
O to a so far unidentified product [
70
].
The possible role of second-sphere active-site amino acids as proton donors and
the role of the Ca
2+
ion close to the active site heme was investigated by compu-
tational chemistry [
67
]. Note that the active site residues His277, Tyr218, and
Arg114 (
W. succinogenes
NrfA numbering) as well as the Ca
2+
ion are strictly
conserved in NrfA enzymes and provide an environment of positive electrostatic
potential around the active site [
71
]. His277 was suggested as the most probable
proton donor, whose spatial orientation and fine-tuned acidity led to energetically
feasible, low-barrier protonation reactions. An alternative candidate was Arg114
according to the theoretical studies by Bykov and Neese [
67
]. Interestingly, Tyr218
did not appear to participate in the reaction during the initial stage of the reduction
process. However, exchange of Tyr218 by phenylalanine led to a more or less
complete loss of nitrite reductase activity, whereas the sulfite reductase activity of
the Tyr218Phe variant remained unaffected [
72
].
Figure 7 The active site of cytochrome
c
nitrite reductase (NrfA) from
Wolinella succinogenes
.
The sulfate molecule (SO
2
4
, from crystallization buffer) occupies the substrate binding site at
heme 1 (see also Figure
5
). The structure emphasizes the set of conserved amino acid residues
including four tyrosine residues Tyr255, Tyr254, Tyr219, and Tyr218 that might play a role in
radical stabilization during catalysis [
69
]. PDB code: 1FS8. This research was originally published
in [
59
] and taken by permission; copyright 2000 American Society for Biochemistry and Molec-
ular Biology.
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