A New Concept for Energy Coupling in Oxidative Phosphorylation Based on a Molecular Explanation of the Oxygen Exchange Reactions

Abstract

The Pi z HOH exchange reaction of oxidative phosphorylation is considerably less sensitive to uncouplers than the Pi T ATP and ATP ;± HOH exchanges. The uncoupler-insensitive Pi 2. HOH Previous findings have shown that the Pi T HOH exchange is less sensitive to 2,4-dinitrophenol than the Pi T. ATP exchange or the capacity for net oxidative l)hosphorylation (10)(11)(12). However, the significance or the source of this exchange has not been known. The possibility exists that it might reflect activities of enzymes such as alkaline phosphatase or pyrophosphatase known to catalyze a Pi ± HOH exchange (7), perhaps activated in some manner by 2,4-dinitrophenol. In addition, whether such behavior is limited to 2,4-dinitrol)henol or might be shown by more potent uncouplers of oxidative phosphorylation has not been shown. Results with other uncouplers and with oligomycin inhibition reported here cover these points.The effects of increasing concentrations of the potent uncoupler 5-chloro-3-tert-butyl-2'-chloro-4'-nitrosalicylanilide (S-13) (13) on the exchanges catalyzed by mitochondria are shown ill Fig. 1. In the absence of uncoupler, the relative rates of the reactions Pi 2. HOH, ATP ¢± HOH, and Pi T ATP are about 12:6: 1, respectively, under the conditions used. At low concentrations of uncoupler, the Pi ± ATP and the ATP z HOH exchanges are much more sensitive to the uncoupler than the Pi z HOH exchange. At a concentration of S-13 sufficient to inhibit the Pi T ATP and ATP 2 HOH exchange by about 50%, the Pi T HOH exchange is inhibited by less than 5%. At a concentration S-13 that gives a near zero value for the Pi T ATP and ATP T HOH exchanges and a maximum value for the uncoupler-stimulated ATPase activity, the Pi T HOH exchange is still rapid and inhibited by only 35%. Responses similar to those reported in Fig. 1 for S-13 with mitochondria are also observed with 2,4-dinitrophenol and m-chlorocarbonvlcvanide phenylhydrazone.Important for the present considerations are the demonstrations, not given in detail here, of the effects of oligomycin.This antibiotic is a potent inhibitor of oxidative phosphorylation and inhibits the Pi T HOH exchange (14). In the absence of uncouplers and under conditions like those described with Fig. 1 Abbreviation: S-13, 5-chloro-3-tert-butyl-2'-chloro-4'-nitrosalicylanilide.

The mechanism of the conservation of energy of biological oxidations

“…In 1973, Boyer [13] and Slater [14] suggested that the energy for ATP synthesis is required not for the esterification of ADP by phosphate per se but for the dissociation of ATP. This postulate is now strongly supported by the following evidence.…”

Energy transduction and proton translocation by adenosine triphosphatases

“…That such conformation changes might be coupled to covalent bond formation in muscle or mitochondria was suggested a decade ago [21]. More recently, the concept has been sharpened to suggest that ATP synthesis is driven by conformationally-linked changes in the affinity for reactants at the catalytic site [ 14,15,22,23]. These affinity change&could readily allow conformational energy to be used for covalent bond formation.…”

Reconstituted mitochondrial oligomycin‐sensitive ATPase (F0F1) with intermediate Pi ⇌ HOH exchange but no Pi ⇌ ATP exchange activity

“…The actual conversion of bound ADP and Pi into ATP is regarded as proceeding without direct coupling to an energy supply. Important evidence suggesting this concept is based on the observation [10][11][12][13][14][15] that submitochondrial particles catalyze an oligomycin-and/or DCCD-sensitive, uncoupler-insensitive exchange of oxygen between Pi and H20 accompanying the formation of Pi from ATP ('intermediate Pi "~ HOH exchange'). Based on detailed studies of this exchange reaction [17,18] an alternating catalytic site mechanism was recently formulated [18], according to which an energy-requiring conformational change of F1 [21 ].…”

“…Evidence accumulated in recent years suggests that the mitochondrial ATPase F~ (and similar ATPases of chloroplasts and bacteria) can undergo energy-linked conformational changes that may be intrinsically involved in ATP synthesis coupled to electron and proton transport [1][2][3][4][5][6][7][8][9][10][11][12][13][14]. It has been proposed [ 12,[15][16][17] that energy transduced through such conformational changes serves to release ATP from [7--17] and to promote productive binding of ADP and Pi to the enzyme [11-14, 17,18].…”