Effects of subunit I mutations on redox-linked conformational changes of the Escherichia coli bo-type ubiquinol oxidase revealed by Fourier-transform infrared spectroscopy.
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.
Department of Biophysics, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto, 606-8502, Japan.
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Y
Yamazaki
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Kandori
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Mogi
D001665:Binding Sites; D003300:Copper / Q000378:metabolism; D003486:Cyanides / Q000378:metabolism; D003573:Cytochrome b Group; D003580:Cytochromes / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D018698:Glutamic Acid; D006418:Heme; D009154:Mutation; D010084:Oxidation-Reduction; D011487:Protein Conformation; D012256:Riboflavin / Q000378:metabolism; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods*; D013057:Spectrum Analysis / Q000379:methods
D003486:Cyanides; D003573:Cytochrome b Group; D003580:Cytochromes; D029968:Escherichia coli Proteins; D018698:Glutamic Acid; D006418:Heme; D003300:Copper; C017168:cytochrome bo, E coli; D012256:Riboflavin