Redox FTIR difference spectroscopy using caged electrons reveals contributions of carboxyl groups to the catalytic mechanism of haem-copper oxidases.

Redox spectra of the haem-copper oxidases cytochrome aa3 of Rhodobacter sphaeroides and cytochrome bo3 of Escherichia coli were recorded in the visible and infrared spectral regions. The reduction of oxidases was initiated after light activation of the 'caged electron' donor riboflavin. Infrared redox difference spectra exhibit absorbance changes in the amide I region, which are indicative of very small redox-linked conformational movements in the polypeptide backbone. A reproducible redox-dependent pattern of positive and negative absorption changes is found in the carbonyl region (1680-1750 cm(-1)). The carbonyl bands shift to lower frequencies due to isotope exchange of the solvent H2O to D2O. This common feature of cytochrome c and quinol oxidases indicates that at least (i) one redox-sensitive carboxyl group is in the protonated state in the oxidized form and (ii) one carboxylic acid is involved at a catalytic step--presumably in proton translocation--of haem-copper oxidase.