Probing protein-cofactor interactions in the terminal oxidases by second derivative spectroscopy: study of bacterial enzymes with cofactor substitutions and heme A model compounds.
Probing protein-cofactor interactions in the terminal oxidases by second derivative spectroscopy: study of bacterial enzymes with cofactor substitutions and heme A model compounds.
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通过二阶导数光谱探测末端氧化酶中的蛋白质-辅因子相互作用：研究具有辅因子取代的细菌酶和血红素 A 模型化合物。
DOI：
10.1002/pro.5560031123
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发表时间：
1994
期刊：
影响因子：
--
通讯作者：
Fujiwara,T
中科院分区：
文献类型：
--
作者：
Felsch,JS;Horvath,MP;Gursky,S;Hobaugh,MR;Goudreau,PN;Fee,JA;Morgan,WT;Admiraal,SJ;Ikeda-Saito,M;Fujiwara,T
Second derivative absorption spectra are reported for theaa3‐cytochromecoxidase from bovine cardiac mitochondria, theaa3‐600 ubiquinol oxidase fromBacillus subtilis, theba3‐cytochromecoxidase fromThermus thermophilis, and theaco‐cytochromecoxidase fromBacillusYN‐2000. Together these enzymes provide a range of cofactor combinations that allow us to unequivocally identify the origin of the 450‐nm absorption band of the terminal oxidases as the 6‐coordinate low‐spin heme, cytochromea.The spectrum of theaco‐cytochromecoxidase further establishes that the split Soret band of cytochromea, with features at 443 and 450 nm, is common to all forms of the enzyme containing ferrocytochromeaand does not depend on ligand occupancy at the other heme cofactor as previously suggested. To test the universality of this Soret band splitting for 6‐coordinate low‐spin heme A systems, we have reconstituted purified heme A with the apo forms of the heme binding proteins, hemopexin, histidine‐proline‐rich glycoprotein and the H64V/V68H double mutant of human myoglobin. All 3 proteins bound the heme A as a (bis)histidine complex, as judged by optical and resonance Raman spectroscopy. In the ferroheme A forms, none of these proteins displayed evidence of Soret band splitting. Heme A‐(bis)imidazole in aqueous detergent solution likewise failed to display Soret band splitting. When the cyanide‐inhibited mixed‐valence form of the bovine enzyme was partially denatured by chemical or thermal means, the split Soret transition of cytochromeacollapsed into a single band at 443 nm. Taken together these data suggest that the observation of Soret splitting, including a feature at 450 nm, results from specific protein‐cofactor interactions that are unique to the cytochromea‐binding pocket of the terminal oxidases. The conservation of this unique binding pocket among evolutionarily distant species may reflect some mechanistic significance for this structure.
