The purpose of the work was to provide a crystallographic demonstration of the venerable idea that CO photolyzed from ferrous heme-a 3 moves to the nearby cuprous ion in the cytochrome c oxidases. Crystal structures of CO-bound cytochrome ba 3-oxidase from Thermus thermophilus, determined at ∼ 2.8-3.2 Å resolution, reveal a Fe-C distance of ~ 2.0 Å, a Cu-O distance of 2.4 Å and a Fe-C-O angle of ~ 126°. Upon photodissociation at 100 K, X-ray structures indicate loss of Fe a3-CO and appearance of Cu B-CO having a Cu-C distance of ~ 1.9 Å and an O-Fe distance of ~ 2.3 Å. Absolute FTIR spectra recorded from single crystals of reduced ba 3-CO that had not been exposed to X-ray radiation, showed several peaks around 1975 cm - 1; after photolysis at 100 K, the absolute FTIR spectra also showed a significant peak at 2050 cm - 1. Analysis of the 'light' minus 'dark' difference spectra showed four very sharp CO stretching bands at 1970 cm - 1, 1977 cm - 1, 1981 cm - 1, and 1985 cm - 1, previously assigned to the Fe a3-CO complex, and a significantly broader CO stretching band centered at ~ 2050 cm - 1, previously assigned to the CO stretching frequency of Cu B bound CO. As expected for light propagating along the tetragonal axis of the P4 32 12 space group, the single crystal spectra exhibit negligible dichroism. Absolute FTIR spectrometry of a CO-laden ba 3 crystal, exposed to an amount of X-ray radiation required to obtain structural data sets before FTIR characterization, showed a significant signal due to photogenerated CO 2 at 2337 cm - 1 and one from traces of CO at 2133 cm - 1; while bands associated with CO bound to either Fe a3 or to Cu B in "light" minus "dark" FTIR difference spectra shifted and broadened in response to X-ray exposure. In spite of considerable radiation damage to the crystals, both X-ray analysis at 2.8 and 3.2 Å and FTIR spectra support the long-held position that photolysis of Fe a3-CO in cytochrome c oxidases leads to significant trapping of the CO on the Cu B atom; Fe a3 and Cu B ligation, at the resolutions reported here, are otherwise unaltered. This article is part of a Special Issue entitled: Respiratory Oxidases.
Bibliographical noteFunding Information:
This study was supported by NIH grant GM35342 (JAF) and NSF grant CHE-1026369 (JTS). Part of this research was carried out at the Stanford Synchrotron Radiation Lightsource, a national user facility operated by Stanford University on behalf of the US Department of Energy, Office of Basic Energy Sciences. The SSRL Structural Molecular Biology Program is supported by the Department of Energy, Office of Biological and Environmental Research, and by the National Institutes of Health, National Center for Research Resources, Biomedical Technology Program, and the National Institute of General Medical Sciences.
- CO photodissociation
- Carbon monoxide
- Cytochrome ba oxidase
- Cytochrome c oxidase
- Fourier transform infrared
- Thermus thermophilus