Evaluation of ligand exchange as a model of heterogeneity within the binuclear core of cytochrome c oxidase.
Dilution of detergent-solubilized cytochrome c oxidase has been found to increase the pH sensitivity of the a3-CuB site. Comparison of a large number of preparations, each purified at pH 8.8, showed either one or two exponential reactivity with cyanide, indicating that pH was not the only factor regulating kinetic heterogeneity within the binuclear core. Differences in heme a concentration during the soluble stages of purification were shown to have a substantial effect on the distribution of both optical and kinetic states within the isolated enzyme. Similar dilution effects were also inducible after purification. In particular, the apparent pKa of the Soret band maximum was shifted to higher pH following dilution of the enzyme. The resulting increase in the 414-nm conformer was also associated with enhanced conversion to the slow cyanide-binding form, suggesting that a single protonation event may give rise to both optical and kinetic changes. Two lines of evidence were contrary to this: First, by appropriate choice of experimental conditions it was possible to observe 30-50% of the cytochrome a3 centers in the 414-nm state with no associated evidence of the slow conformer. Second, the addition of formate at pH 8.8 induced conversion to both slow and 414-nm conformers, but the rates and extents of conversion were not correlated. Separate structural events are therefore proposed to modulate the optical and kinetic properties of the binuclear core. Since acidic pH has the well-established ability to induce both rapid-->slow and 430-->414-nm conversions, we suggest that the effects of dilution and formate are to mimic an acid jump by facilitating proton uptake into the binuclear core.
Gullo
S M
SM
Department of Chemistry, Northern Illinois University, DeKalb 60115.
Tayh
J A
JA
Li
J
J
Baker
G M
GM
D000818:Animals; D002417:Cattle; D003486:Cyanides / Q000378:metabolism; D003576:Electron Transport Complex IV / Q000302:isolation & purification / Q000378:metabolism*; D005561:Formates / Q000378:metabolism / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008024:Ligands; D008929:Mitochondria, Heart / Q000201:enzymology; D013053:Spectrophotometry; D013997:Time Factors
D003486:Cyanides; D005561:Formates; D008024:Ligands; D003576:Electron Transport Complex IV