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.
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 (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 nu(NO) modes from the light-induced FTIR difference spectra are isolated from other perturbed vibrations using 15NO and 15N18O. The nu(N-O)a3 is observed at 1622 cm-1, and upon photolysis, it is replaced by a new nu(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 nu(NO) to an O-bound (eta1-O) or a side-on (eta2-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 CuB-NO species; the [{FeNO}7{CuNO}11] complex is suggested here to be an inhibitory complex.
Hayashi
Takahiro
T
Department of Molecular Biology, The Scripps Research Institute, La Jolla, California 92037, USA.
Lin
I-Jin
IJ
Chen
Ying
Y
Fee
James A
JA
Moënne-Loccoz
Pierre
P
D003573:Cytochrome b Group / Q000737:chemistry* / Q000378:metabolism*; D003576:Electron Transport Complex IV / Q000737:chemistry* / Q000378:metabolism*; D006418:Heme / Q000737:chemistry* / Q000378:metabolism; D010088:Oxidoreductases / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D017550:Spectroscopy, Fourier Transform Infrared; D016963:Thermus thermophilus / Q000201:enzymology*
D003573:Cytochrome b Group; D006418:Heme; D010088:Oxidoreductases; C058557:cytochrome ba3; C024748:copper oxidase; C034747:nitric-oxide reductase; D003576:Electron Transport Complex IV