Structural changes that occur upon photolysis of the Fe(II) a3-CO complex in the cytochrome ba 3-oxidase of Thermus thermophilus: A combined X-ray crystallographic and infrared spectral study demonstrates CO binding to Cu B

摘要

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 ? resolution, reveal a Fe-C distance of ~ 2.0 ?, a Cu-O distance of 2.4 ? 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 ? and an O-Fe distance of ~ 2.3 ?. 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 ? 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.