Fourier Transform Infrared Characterization of a Cu_B-Nitrosyl Complex in Cytochrome ba_3 from Thermus thermophilus: Relevance to NO Reductase Activity in Heme-Copper Terminal Oxidases

摘要

The two heme-copper terminal oxidases of Thermus thermophilus have been shown to catalyze the two-electron reduction of nitric oxide (NO) to nitrous oxide (N_2O) [Giuffre, A.; Stubauer, G.; Sarti, P.; Brunori, M.; Zumft, W. G.; Buse, G.; Soulimane, T. Proc. Natl. Acad. Sci. U.S.A. 1999, 96,14718-14723]. While it is well-established that NO binds to the reduced heme a_3to form a low-spin heme {FeNO}7 species, the role Cu_b plays in the binding of the second NO remains unclear. Here we present low-temperature FTIR photolysis experiments carried out on the NO complex formed by addition of NO to fully reduced cytochrome ba_3. Low-temperature UV-vis, EPR, and RR spectroscopies confirm the binding of NO to the heme a_3 and the efficiency of the photolysis at 30 K. The v(NO) modes from the light-induced FTIR difference spectra are isolated from other perturbed vibrations using ~(15)NO and ~(15)N~(18)O. The v(N-O)a_3 is observed at 1622 cm~(-1), and upon photolysis, it is replaced by a new v(N-O) at 1589 cm~(-1) assigned to a CuB-nitrosyl complex. This N-O stretching frequency is more than 100 cm"1 lower than those reported for Cu-NO models with three N-ligands and for Cu_b~+-NO in bovine aa_3. Because the UV-vis and RR data do not support a bridging configuration between Cub and heme a3 for the photolyzed NO, we assign the exceptionally low v(NO) to an O-bound (η~1-O) or or a side-on (η~2-NO) Cu_B-nitrosyl complex. From this study, we propose that, after binding of a first NO molecule to the heme a3 of fully reduced Tt ba_3, the formation of an N-bound {CuNO}~(11) is prevented, and the addition of a second NO produces an O-bond Cus-hyponitrite species bridging Cub and Fea_3. In contrast, bovine cytochrome c oxidase is believed to form an N-bound Cub-NO species; the [{FeNO}~7{CuNO}~(11)] complex is suggested here to be an inhibitory complex.