GLUTAMATE-286 MUTANTS OF CYTOCHROME BO-TYPE UBIQUINOL OXIDASE FROM ESCHERICHIA-COLI - INFLUENCE OF MUTATIONS ON THE BINUCLEAR CENTER STRUCTURE REVEALED BY FT-IR AND EPR SPECTROSCOPIES

Glutamate-286 mutants of cytochrome be-type ubiquinol oxidase from Esc herichia cell were examined by EPR and FT-IR spectroscopies, We confir med a very low enzymatic activity for E286Q, However, E286D retained o ne-third of the wild-type activity, probably due to the presence of th e carboxylic group on the side-chain, The effect of the mutations at p osition 286 on the binuclear site was observed clearly in the EPR spec tral change for the air-oxidized state, The effect was more significan tly manifested in the presence of cyanide or azide in the oxidized sta te, In contrast, the mutations only slightly perturbed the binuclear c enter of the GO-reduced enzymes, These results indicate the importance of a direct through-bond connectivity between Gu(B) and Glu(286) via Pro(285) and His(284). (C) 1997 Federation of European Biochemical Soc ieties.