Pozzan T, Di Virgilio F, Bragadin M, Miconi V, Azzone G F
Proc Natl Acad Sci U S A. 1979 May;76(5):2123-7. doi: 10.1073/pnas.76.5.2123.
H(+)/site, charge/site, and ATP/site ratios have been determined at coupling sites I, II, and III. Three e(-) donors have been used for coupling site III: ferrocyanide, ascorbate + tetramethyl-p-phenylenediamine (TMPD), and succinate + TMPD. The H(+)/site ratios are 4.0 with ferrocyanide and 6.0 with succinate + TMPD (at pH <7.0); the charge/site ratios are 6.0 with ferrocyanide and with succinate + TMPD (at pH <7.0) and 4.0 with ascorbate + TMPD; the ATP/site ratio is 1.34 with ascorbate + ferrocyanide. These ratios have been obtained in the presence of amounts of antimycin A that provide full inhibition of site II. For coupling sites I and II, ferricyanide has been used as e(-) acceptor and succinate or NAD-linked substrates as e(-) donors. The H(+)/site ratios are 4.0 at sites I and II; the charge/site ratios are 4.0 at site I and 2.0 at site II; the ATP/site ratios are 1.0 at site I and 0.5 at site II. Two major factors affect the stoichiometries: (i) dimension of unk and (ii) supply of H(+) from the matrix. There is a correlation between collapse of unk and increase of H(+)/site and charge/site ratios. This indicates that approximation of the phenomenologic stoichiometry of the H(+) pump is obtained when flow ratios are measured at level flow. That charge/site and ATP/site ratios increase when ferrocyanide is e(-) donor and decrease when ferricyanide is e(-) acceptor is attributed to the localization of the redox couple. This leads to separation of 1 charge/e(-) when ferrocyanide is e(-) donor and to consumption of 1 charge/e(-) when ferricyanide is e(-) acceptor. To account for an extrusion of H(+) in excess of that predicted by the loop model, it is proposed that each coupling site contains a channel acting as a H(+) pump.
已测定了偶联位点I、II和III处的H⁺/位点、电荷/位点和ATP/位点比率。已使用三种电子供体用于偶联位点III：亚铁氰化物、抗坏血酸 + 四甲基 - 对苯二胺（TMPD）以及琥珀酸 + TMPD。亚铁氰化物的H⁺/位点比率为4.0，琥珀酸 + TMPD的H⁺/位点比率为6.0（在pH < 7.0时）；亚铁氰化物和琥珀酸 + TMPD的电荷/位点比率为6.0（在pH < 7.0时），抗坏血酸 + TMPD的电荷/位点比率为4.0；抗坏血酸 + 亚铁氰化物的ATP/位点比率为1.34。这些比率是在存在能完全抑制位点II的抗霉素A的情况下获得的。对于偶联位点I和II，铁氰化物用作电子受体，琥珀酸或NAD连接的底物用作电子供体。位点I和II处的H⁺/位点比率为4.0；位点I处的电荷/位点比率为4.0，位点II处为2.0；位点I处的ATP/位点比率为1.0，位点II处为0.5。有两个主要因素影响化学计量关系：（i）未知的维度和（ii）来自基质的H⁺供应。未知的崩溃与H⁺/位点和电荷/位点比率的增加之间存在相关性。这表明当在水平流处测量流量比率时，可得到H⁺泵的现象学化学计量关系的近似值。当亚铁氰化物为电子供体时电荷/位点和ATP/位点比率增加，而当铁氰化物为电子受体时则降低，这归因于氧化还原对的定位。这导致当亚铁氰化物为电子供体时每电子有1个电荷分离，而当铁氰化物为电子受体时每电子消耗1个电荷。为了解释H⁺的挤出量超过环模型预测的量，有人提出每个偶联位点包含一个充当H⁺泵的通道。
