H+/site, charge/site, and ATP/site ratios at coupling site IIi in mitochondrial electron transport

Azzone, G.F.; Pozzan, T.; Di Virgilio, F.

Journal of Biological Chemistry 254(20): 10206-10212

1979


ISSN/ISBN: 0021-9258
PMID: 39933
Accession: 068518722

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Abstract
Proton/site, charge/site, and ADP/site stoichiometries were determined at coupling site III in antimycin A-treated rat liver mitochondria. Three e- (electron) donors were used including ferrocyanide, ascorbate plus tetramethyl-p-phenylenediamine (TMPD) and succinate plus TMPD. With ferrocyanide as e- donor the charge/site ratio is 5.0 to 6.0 and the ADP/site ratio 1.3; the H+/site ratio is about 4.0. Lower ratios are observed when ferrocyanide is added to valinomycin-treated mitochondria as compared to when valinomycin is added to ferrocyanide-treated mitochondria. With ascorbate plus TMPD as e- donor the charge/site ratio approaches 4.0 and the ADP/site ratio 1.1. With succinate plus TMPD as e- donor both the H+/site and the charge/site ratios are about 6.0 at acidic pH and tend to decline at alkaline pH. In these measurements, operation of site II was abolished by 30-40 pmol of antimycin A .times. mg of protein-1 or 600 to 1000 pmol of 2-heptyl-4-hydroxyquinoline-N-oxide (HQNO) .times. mg of protein-1. In these amounts both antimycin A and HQNO inhibit H+ extrusion and cation uptake due to the operation of site II. Higher amounts of antimycin A or HQNO also affect markedly energy conservation as indicated by increase of state 4 respiration and collapse of .DELTA.psi. The mitochondrial respiratory chain may contain at coupling site III a H+ pump the operation of which leads to the extrusion of 4 H+ and to the separation of 4 charges. The additional separation of 2 charges is attributed to the vectorial transfer of 2 e- through cytochrome oxidase.