Q-Band ENDOR (Electron Nuclear Double Resonance) of the Heme o3 Liganding Environment at the Binuclear Center in Cytochrome bo3 from Escherichia coli

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• 作者：Andrei V. Veselov ; Jeffrey P. Osborne ; Robert B. Gennis ; and Charles P. Scholes
• 刊名：Journal of the American Chemical Society
• 出版年：2000
• 出版时间：September 13, 2000
• 年：2000
• 卷：122
• 期：36
• 页码：8712 - 8716
• 全文大小：59K
• 年卷期：v.122,no.36(September 13, 2000)
• ISSN：1520-5126

文摘

The liganding environment and electronic structure of high-spin ferric heme o3 in the binuclearcenter of bo3 ubiquinol oxidase were probed with Q-band (34.1 GHz) ENDOR. We studied forms of theenzyme where reduction eliminated antiferromagnetic coupling to the nearby Cu_B center. ENDOR comparisonswere made to ¹⁴N heme and histidine nitrogen features, to exchangeable proton features, and to the ¹⁷O-waterfeature of aquometmyoglobin, a high-spin ferric heme protein with a known axial water ligand. Nitrogenfeatures observed from heme and proximal histidine of cytochrome o3 occurred in the range of frequencieswhere they had previously been observed for aquometmyoglobin. However, the proximal histidine of cytochromeo3 was notable in revealing more disorder and a wider range in its hyperfine couplings than inaquometmyoglobin. Di-oxygen-induced turnover of the bo3 enzyme altered both the heme and histidineelectronic structure so as to show after turnover a simpler, better resolved heme and histidine pattern withgreater similarity to the pattern found in aquometmyoglobin. We saw no evidence from cytochrome o3 for the6 MHz exchangeable water proton coupling and the 17.5 MHz ¹⁷O-water coupling exhibited by aquometmyoglobin. A plausible conclusion from such a negative result is that the high-spin ferric o3 heme which westudied has no covalently attached axial sixth OH_X ligand when magnetically decoupled from Cu_B. Comparisonof cytochrome o3 in protonated and deuterated solvents definitively indicated no exchangeable proton couplingsgreater than 3.5 MHz. An implication of our study is that in the magnetically decoupled high-spin ferriccytochrome o3 there is either no sixth OH_X ligand or, if there is any "sixth" OH_X ligand to cytochrome o3 thatcan exchange with ¹⁷O-water, it would have to be off-axis, disordered, and weakly liganded to the heme.