Fourier transform infrared characterization of a CuB-nitrosyl complex in cytochrome ba3 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 2-electron reduction of nitric oxide (NO) to nitrous oxide (N2O) [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 a3 to form a low-spin heme {FeNO}⁷ species, the role CuB 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 ba3. Low-temperature UV-vis, EPR, and RR spectroscopies confirm the binding of NO to the heme a3 and the efficiency of the photolysis at 30 K. The ν(NO) modes from the light-induced FTIR difference spectra are isolated from other perturbed vibrations using ¹⁵NO and ¹⁵N¹⁸O. The ν(N-O)a3 is observed at 1622 cm⁻¹, and upon photolysis, it is replaced by a new ν(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 CuB ⁺-NO in bovine aa3. 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 ν(NO) to an O-bound (η¹-O) or a side-on (η²-NO) CuB-nitrosyl complex. From this study, we propose that, after binding of a first NO molecule to the heme a3 of fully-reduced Tt ba3, the formation of an N-bound {CuNO}¹¹ is prevented, and the addition of a second NO produces an O-bond CuB-hyponitrite species bridging CuB and Fea₃. In contrast, bovine cytochrome c oxidase is believed to form an N-bound Cu_B-NO species; the [{FeNO}⁷{CuNO}¹¹] complex is suggested here to be an inhibitory complex.