pH effect on the Soret absorption band of resting cytochrome c oxidase
Walter, Scott Allen
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The pH dependance of the near-uv absorption band of oxidized (Fe⁺³) cytochrome c oxidase has been investigated. Decreasing the pH of an oxidase sample from 8.9 to 6.5 caused a progressive blue shift from 422-424 nm to approximately 417 nm, while titration back to high pH (10.5) led to a reversal of the Soret to 426 nm. The time required for the band to reach a stable, equilibrium position ranged from 30 minutes to 8 hours. The extent of shift was also highly variable from one preparation to the next, as well as from one experiment to another within the same preparation. An isosbestic point was present in most cases, typically near 421 nm during blue shifts, and 425 nm during red shifts. The kinetic approach to the equilibrium position was measured both for blue and red shifting enzyme samples. The shift in both directions was characterized by two phases of reactivity which had observed rate constants (k[sub obs]) differing by two orders of magnitude (10⁻² and 10⁻⁴ s⁻¹). The amplitude of the fast phase ranged from 30% to 75% of the total absorbance change. The direction of the shift had no effect on the rate constants associated with the fast and slow phases. Additionally, the blue shift of the Soret band was not kinetically correlated to the fast to slow conversion in ligand binding behavior which is also known to occur at low pH. Resistent samples which showed little or no response to low pH often failed to have an isosbestic point, and were often associated with a red shift of the Soret band after 1-2 hours at low pH. These effects are all due to cytochrome a₃, not cytochrome a. Some samples which were incubated at high pH (>9.1) exhibited a loss of amplitude which was quickly reversible pH (> 9.1) exhibited a loss of amplitude which was quickly reversible after titration to low pH, and caused the spectrum before titration to deviate from the isosbestic point of the spectra recorded after titration. This probably reflects an effect on cytochrome a (Baker & Palmer, 1987), and did not affect pH sensitivity. Incubation of resistent preparations at high pH and high concentrations of dodecyl maltoside caused a marked increase in pH sensitivity. The kinetics of pH resistant samples showed the fast component described above, but displayed little or no slow phase. Incubation at high concentrations of dodecyl maltoside restored the biphasic response to low pH, resulting in a greater blue shift. High concentrations of cholate during isolation and purification of the enzyme did not affect pH sensitivity, although it did increase the amount of bound phospholipid by as much as ten times that present when the enzyme was isolated using the minimum amount of cholate needed to extract oxidase from mitochondrial membranes. A model consistent with the fast and slow proton binding forms is described.