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Proton Delivery in NO Reduction by Fungal Nitric-oxide Reductase

2000; Elsevier BV; Volume: 275; Issue: 7 Linguagem: Inglês

10.1074/jbc.275.7.4816

1083-351X

Hideaki Shimizu, E. Obayashi, Yuki Gomi, Hiroshi Arakawa, Sam‐Yong Park, Hiro Nakamura, Shin‐ichi Adachi, Hirofumi Shoun, Yoshitsugu Shiro,

Photosynthetic Processes and Mechanisms

Fungal nitric-oxide reductase (NOR) is a heme enzyme that catalyzes the reduction of NO to N 2 O through its ferric-NO complex, the first intermediate of 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 enzymes were determined to 1.7-Å 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 hemoproteins. In the WT structure, a specific hydrogen-bonding network that connects the active site to the solvent was identified, H 2 O(Wat 74 )-Ser 286 -H 2 O(Wat 33 )-Asp 393 -solvent. Wat 74 is located 3.10 Å from the iron-bound NO. Replacement of Ser 286 with Val or Thr scarcely alters the NO coordination structure but expels the water molecules, Wat 74 from the active site. The Asp 393 mutation does not influence the position of Wat 74 , but disrupts the hydrogen-bonding network at Wat 33 , as evidenced by enzymatic, kinetic, and spectroscopic (resonance Raman and IR) results. The structural changes observed upon the Ser 286 or the Asp 393 mutation are consistent with the dramatic loss of the enzymatic activity for the NO reduction of 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 addition, we find the hydrogen-bonding network, H 2 O(Wat 74 )-Ser 286 -H 2 O(Wat 33 )-Asp 393 , as a proton delivery pathway in the NO reduction reaction by fungal NOR.