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

J Am Chem Soc. 2007 Dec 5;129(48):14952-8. doi: 10.1021/ja074600a. Epub 2007 Nov 13.

Abstract

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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Cytochrome b Group / chemistry*
  • Cytochrome b Group / metabolism*
  • Electron Transport Complex IV / chemistry*
  • Electron Transport Complex IV / metabolism*
  • Heme / chemistry*
  • Heme / metabolism
  • Oxidoreductases / chemistry*
  • Oxidoreductases / metabolism*
  • Protein Binding
  • Spectroscopy, Fourier Transform Infrared
  • Thermus thermophilus / enzymology*

Substances

  • Cytochrome b Group
  • Heme
  • Oxidoreductases
  • cytochrome ba3
  • copper oxidase
  • nitric-oxide reductase
  • Electron Transport Complex IV