Effects of subunit I mutations on redox-linked conformational changes of the Escherichia coli bo-type ubiquinol oxidase revealed by Fourier-transforminfrared spectroscopy

Cytochrome be is the heme-copper terminal ubiquinol oxidase in the aerobic respiratory chain of Escherichia cell, and functions as a redox-coupled pro ton pump. As an extension to our mutagenesis and Fourier-transform infrared studies on ion pumps, we examined the effects of subunit I mutations on re dox-linked protein structural changes in cytochrome be, Upon photo-reductio n in the presence of riboflavin, Y288F and H333A showed profound effects in their peptide backbone vibrations (amide-I and amide-II), probably due to the loss of Cu, or replacement of high-spin heme o with heme B, In the freq uency region of protonated carboxylic C=O stretching vibrations, negative 1 ,743 cm(-1) and positive 1,720 cm(-1) bands were observed in the wild-type; the former shifted to 1,741 cm(-1) in E286D but not in other mutants inclu ding D135N. This suggests that Glu286 in the D-channel is protonated in the air-oxidized state and undergoes hydrogen bonding changes upon reduction o f the redox metal centers, Two pairs of band shifts at 2,566 (+)/2,574 (-) and 2,546 (+)/2,556 (-) cm(-1) in all mutants indicate that two cysteine re sidues not in the vicinity of the metal centers undergo redox-linked hydrog en bonding changes. Cyanide had no effect on the protein structural changes because of the rigid local protein structure around the binuclear center o r the presence of a ligand(s) at the binuclear center, and was released fro m the binuclear center upon reduction. This study establishes that cytochro me be undergoes unique redox-linked protein structural changes. Localizatio n and time-resolved analysis of the structural changes during dioxygen redu ction will facilitate understanding of the molecular mechanism of redox-cou pled proton pumping at the atomic level.