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- Title
Effects of Subunit I Mutations on Redox-Linked Conformational Changes of the Escherichia coli bo-type Ubiquinol Oxidase Revealed by Fourier-Transform Infrared Spectroscopy1.
- Authors
Yamazaki, Yoichi; Kandori, Hideki; Mogi, Tatsushi
- Abstract
Cytochrome bo is the heme-copper terminal ubiquinol oxidase in the aerobic respiratory chain of Escherichia coli, and functions as a redox-coupled proton pump. As an extension to our mutagenesis and Fourier-transform infrared studies on ion pumps, we examined the effects of subunit I mutations on redox-linked protein structural changes in cytochrome bo. Upon photo-reduction 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 CuB or replacement of high-spin heme o with heme B. In the frequency 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 including D135N. This suggests that Glu286 in the D-channel is protonated in the air-oxidized state and undergoes hydrogen bonding changes upon reduction of 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 residues not in the vicinity of the metal centers undergo redox-linked hydrogen bonding changes. Cyanide had no effect on the protein structural changes because of the rigid local protein structure around the binuclear center or the presence of a ligand(s) at the binuclear center, and was released from the binuclear center upon reduction. This study establishes that cytochrome bo undergoes unique redox-linked protein structural changes. Localization and time-resolved analysis of the structural changes during dioxygen reduction will facilitate understanding of the molecular mechanism of redox-coupled proton pumping at the atomic level.
- Subjects
GENETIC mutation; UBIQUINOL-cytochrome c reductase; PORPHYRINS; RADIOGENETICS; TETRAPYRROLES
- Publication
Journal of Biochemistry, 1999, Vol 126, Issue 1, p194
- ISSN
0021-924X
- Publication type
Article
- DOI
10.1093/oxfordjournals.jbchem.a022423