Detection of the His-Heme Fe2+−NO Species in the Reduction of NO to N2O byba3-Oxidase fromThermusthermophilus

Abstract

Reaction pathways in the enzymatic formation and cleavage of the N−N and N−O bonds, respectively, are difficult to verify without the structure of the intermediates, but we now have such information on the heme a₃²⁺-NO species formed in the reaction of ba₃-oxidase with NO from resonance Raman spectroscopy. We have identified the His-heme a₃²⁺-NO/Cu_B¹⁺ species by its characteristic Fe−NO and N−O stretching frequencies at 539 and 1620 cm^-1, respectively. The Fe−NO and N−O frequencies in ba₃-oxidase are 21 and 7 cm^-1 lower and higher, respectively, than those observed in Mb−NO. From these results and earlier Raman and FTIR measurements, we demonstrate that the protein environment of the proximal His384 that is part of the Q-proton pathway controls the strength of the Fe−His384 bond upon ligand (CO vs NO) binding. We also show by time-resolved FTIR spectroscopy that Cu_B¹⁺ has a much lower affinity for NO than for CO. We suggest that the reduction of NO to N₂O by ba₃-oxidase proceeds by the fast binding of the first NO molecule to heme a₃ with high-affinity, and the second NO molecule binds to Cu_B with low-affinity, producing the temporal co-presence of two NO molecules in the heme-copper center. The low-affinity of Cu_B for NO binding also explains the NO reductase activity of the ba₃-oxidase as opposed to other heme-copper oxidases. With the identification of the His-heme a₃²⁺-NO/Cu_B¹⁺ species, the structure of the binuclear heme a₃-Cu_B¹⁺ center in the initial step of the NO reduction mechanism is known.