Detection of the his-heme Fe2+-NO species in the reduction of NO to N2O by ba3-oxidase from Thermus thermophilus

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 a 3 2+ -NO species formed in the reaction of ba 3 -oxidase with NO from resonance Raman spectroscopy. We have identified the His-heme a 3 2+ -NO/Cu B 1+ 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 ba 3 -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 B 1+ has a much lower affinity for NO than for CO. We suggest that the reduction of NO to N 2 O by ba 3 -oxidase proceeds by the fast binding of the first NO molecule to heme a 3 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 ba 3 -oxidase as opposed to other heme-copper oxidases. With the identification of the His-heme a 3 2+ -NO/Cu B 1+ species, the structure of the binuclear heme a 3 -Cu B 1+ center in the initial step of the NO reduction mechanism is known.