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

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.