Proton delivery in NO reduction by fungal nitric-oxide reductase - Cryogenic crystallography, spectroscopy, and kinetics of ferric-NO complexes of wild-type and mutant enzymes

Fungal nitric-oxide reductase (NOR) is a heme enzyme that catalyzes the red uction of NO to N2O through its ferric-NO complex, the first intermediate o f the catalysis. Crystal structures of the ferric-NO forms of wild type (WT ) fungal NOR, and of the Ser(286) --> Val and Ser(286) --> Thr mutant enzym es were determined to 1.7-Angstrom resolution at cryogenic temperature (100 K). This shows a slightly tilted and bent NO binding to the heme iron, in sharp contrast to the highly bent NO coordination found in ferrous hemoprot eins, In the WT structure, a specific hydrogen-bonding network that connect s the active site to the solvent was identified, H2O(Wat(74))-Ser(286)-H2O- (Wat(33))-Asp(393)-solvent. Wat(74) is located 3.10 Angstrom from the iron -bound NO. Replacement of Ser(286) With Val or Thr scarcely alters the NO c oordination structure but expels the water molecules, Wat(74) from the acti ve site. The Asp(393) mutation does not influence the position of Wat74 but disrupts the hydrogen-bonding network at Wat(33), as evidenced by enzymati c, kinetic, and spectroscopic (resonance Raman and IR) results. The structu ral changes observed upon the Ser(286) or the Asp(393) mutation are consist ent with the dramatic loss of the enzymatic activity for the NO reduction o f fungal NOR. We have conclusively identified the water molecule, Wat(74), adjacent to the iron-bound NO as a proton donor to the Fe-NO moiety. In add ition, we find the hydrogen-bonding network, H2O(Wat(74))-Ser-(286)-H2O(Wat (33))-Asp(393), as a proton delivery pathway in the NO reduction reaction b y fungal NOR.