Oxidative titrations of reduced cytochrome aa3: correlation of midpoint potentials and extinction coefficients observed at three major absorption bands

Abstract

Anaerobic oxidative titrations of purified cytochrome aa3 were monitored at three wavelengths (444, 604, and 820 nm), in both the absence and the presence of carbon monoxide. Computer simulation of each titration curve was utilized to ascertain the midpoint potentials of the four oxidation-reduction centers of the enzyme. For experiments performed under nitrogen, two components were found to titrate with low potential (heme aL = 220 mV, CuL = 240 mV) and two with high potential (heme ath, cuH = 340 mV), consistent with results obtained previously in reductive titrations. Unequal heme extinction coefficients were observed at 444 nm. Oxidation by either potassium ferricyanide or 1,1'-bis(hydroxymethyl)ferricinium ion showed that the low potential heme component contributed 75% of the absorbance change at 444 nm. At 820 nm, the entire absorbance change could be attributed to a single, low potential copper component. Midpoint potentials calculated for the carbon monoxide complexed enzyme agreed with previously reported values. The copper components retained the values observed under nitrogen, while the titratable heme group gave an apparent midpoint potential of 260 mV. These results enable us to assign absorbance changes at various wavelengths to specific redox components of cytochrome aa3.