ANTIMYCIN INHIBITS QE-QUENCHING BY A PROTONOPHORIC MECHANISM


Christine T. Yerkes and Antony R. Crofts, Program in Biophysics and Computational Biology and Dept. of Microbiology, University of Illinois, Urbana, IL 61801

We have investigated the inhibition of qE-quenching by antimycin and DCCD, and the enhancement by pre-illumination to form de-epoxidation products. We have shown that i) the effects of antimycin on qE-quenching can all be mimicked by classical uncoupling agents at low concentration; ii) the antimycin titre is dependent on rate of generation of the quenched state (a substantial literature has previously demonstrated that antimycin is an uncoupler, with a concentration dependence which varies with the rate of electron transport); iii) valinomycin potentiates the effect of antimycin in a synergistic and K+-dependent fashion. We conclude that antimycin probably inhibits by a protonophoric mechanism. While most of the above effects seem to reflect a simple protonophoric mechanism, we have observed that the zeaxanthin-enhanced fraction of qE-quenching, and that associated with non-cyclic flow, are more sensitive to antimycin than that driven by an artificial cyclic system, even when account is taken of the different rates of onset. This may reflect a gradient in H+ activity between site at which antimycin acts on the qE-quenching mechanism and the bulk phase. DCCD inhibits qE-quenching after preincubation, but does not uncouple, and likely reflects binding at carboxylic acid resodues buried in the hydrophobic phase. The relation between qE-quenching, aggregation, and light-scattering changes are not simple; quenching occurs in chloroplasts in the absence of Mg2+ (in contrast to earlier results), or after "clipping" by trypsin treatment so as to eliminate the Mg2+ effect on fluorescence; and in media containing 50-100 mM Na-acetate, a delayed qE-quenching and enhanced light-scattering showed no kinetic correlation.

We will discuss the mechanism of qE-quenching in terms of a model suggested by Crofts and Yerkes (1994, FEBS Lett. 352, 265-270). The main elements of the model are: a) The qE-quenching reflects a mechanism associated with a component of the light-harvesting antenna rather than the reaction center of photosystem (PS) II; we suggest that it occurs through formation of an efficient quencher in one of the minor chlorophyll protein (CP) complexes. b) The minor CPs have glutamate residues instead of glutamines at positions shown in light harvesting complex II (LHCII) to be ligands to chlorophylls near the lumenal interface. We suggest that the quenching reflects a change in ligation of chlorophyll on protonation of these glutamate residues leading to formation of an exciton coupled dimer with a neighboring pigment, in which additional energy levels allow vibrational relaxation of the excited singlet. The model accounts for the dependence on low lumenal pH, the ligand residue changes between LHCII and the minor CPs, the preferential distribution of components of the xanthophyll cycle in the minor CPs, the inhibition of qE-quenching by DCCD, and the specific binding of DCCD to the minor CPs.