Structural changes that occur upon photolysis of the Fe(II)_a3-CO complex in the cytochrome ba₃-oxidase of Thermus thermophilus: A combined X-ray crystallographic and infrared spectral study demonstrates CO binding to Cu_B

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• 作者：Bin Liu^a ; ¹ ; Yang Zhang^b ; J. Timothy Sage^b ; ^{jtsage@neu.edu} ; S. Michael Soltis^c ; ^{soltis@slac.stanford.edu} ; Tzanko Doukov^c ; Ying Chen^a ; C. David Stout^a ; ^{dave@scripps.edu} ; James A. Fee^a ; ^{jafee@scripps.edu}
• 关键词：FTIR ; Fourier transform infrared ; MGy ; 1  ; × ;   ; 106 Gray ; FWHM ; full width at half maximum
• 刊名：Biochimica et Biophysica Acta (BBA)/Bioenergetics
• 出版年：2012
• 期刊代码：13_00052728
• 类别：bio
• 出版时间：April, 2012
• 卷：1817
• 期：4
• 页码：658-665
• 文件大小：773 K

摘要

The purpose of the work was to provide a crystallographic demonstration of the venerable idea that CO photolyzed from ferrous heme-a₃ moves to the nearby cuprous ion in the cytochrome c oxidases. Crystal structures of CO-bound cytochrome ba₃-oxidase from Thermus thermophilus, determined at ~ 2.8-3.2 脜 resolution, reveal a Fe-C distance of ~ 2.0 脜, a Cu-O distance of 2.4 脜 and a Fe-C-O angle of ~ 126掳. 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 脜 and an O-Fe distance of ~ 2.3 脜. Absolute FTIR spectra recorded from single crystals of reduced ba₃-CO that had not been exposed to X-ray radiation, showed several peaks around 1975 cm^鈭?#xA0;1; after photolysis at 100 K, the absolute FTIR spectra also showed a significant peak at 2050 cm^鈭?#xA0;1. Analysis of the 鈥榣ight鈥?minus 鈥榙ark鈥?difference spectra showed four very sharp CO stretching bands at 1970 cm^鈭?#xA0;1, 1977 cm^鈭?#xA0;1, 1981 cm^鈭?#xA0;1, and 1985 cm^鈭?#xA0;1, previously assigned to the Fe_a3-CO complex, and a significantly broader CO stretching band centered at ~ 2050 cm^鈭?#xA0;1, previously assigned to the CO stretching frequency of Cu_B bound CO. As expected for light propagating along the tetragonal axis of the P4₃2₁2 space group, the single crystal spectra exhibit negligible dichroism. Absolute FTIR spectrometry of a CO-laden ba₃ 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 CO₂ at 2337 cm^鈭?#xA0;1 and one from traces of CO at 2133 cm^鈭?#xA0;1; while bands associated with CO bound to either Fe_a3 or to Cu_B in 鈥渓ight鈥?minus 鈥渄ark鈥?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 脜 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.