Inhibitor "double occupancy" in the Q(o) pocket of the chloroplast cytochrome b6f complex.
Electron paramagnetic resonance (EPR) spectra of the "Rieske" 2Fe-2S cluster revealed that two molecules of the inhibitor 2,5-dibromo-3-methyl-6-isopropylbenzoquinone (DBMIB) can bind to each monomer of the spinach cytochrome (cyt) b6f complex, both in isolated form and in intact thylakoid membranes. Binding to the high-affinity site, which accounts for the observed inhibitory effects, caused small shifts in the g(x) transition of the 2Fe-2S cluster EPR spectrum, similar to those induced by stigmatellin or 2-iodo-6-isopropyl-3-methyl-2',4,4'-trinitrodiphenyl ether (DNP-INT). Occupancy of the low-affinity site was only observed after addition of superstoichiometric amounts of the inhibitor and was accompanied by the appearance of a g = 1.94 EPR signal. The shape of the equilibrium binding titration curve, the effects on the 2Fe-2S EPR spectrum, and the ability of the DBMIB binding to displace DNP-INT were consistent with two molecules of DBMIB binding at the Q(o) pocket, with the strongly binding species binding close to the 2Fe-2S cluster. Possible implications of these findings for so-called "double-occupancy" models for Q(o) site catalysis are discussed.
Roberts
A G
AG
Institute of Biological Chemistry, Washington State University, 289 Clark Hall, Pullman, Washington 99164-6340, USA.
Kramer
D M
DM
D002384:Catalysis; D002736:Chloroplasts / Q000737:chemistry / Q000201:enzymology*; D003573:Cytochrome b Group / Q000037:antagonists & inhibitors* / Q000737:chemistry; D045346:Cytochrome b6f Complex; D003991:Dibromothymoquinone / Q000737:chemistry / Q000494:pharmacology*; D004578:Electron Spin Resonance Spectroscopy; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D011487:Protein Conformation; D018724:Spinacia oleracea; D014301:Trinitrobenzenes / Q000737:chemistry / Q000494:pharmacology
D003573:Cytochrome b Group; D004791:Enzyme Inhibitors; D014301:Trinitrobenzenes; D003991:Dibromothymoquinone; C026642:2-iodo-6-isopropyl-3-methyl-2',4,4'-trinitrodiphenyl ether; D045346:Cytochrome b6f Complex