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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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