Gaballo A, Zanotti F, Solimeo A, Papa S
Institute of Medical Biochemistry and Chemistry, University of Bari, Italy.
Biochemistry. 1998 Dec 15;37(50):17519-26. doi: 10.1021/bi981422c.
Diamide treatment of the F0F1-ATP synthase in "inside out" submitochondrial particles (ESMP) in the absence of a respiratory Delta mu H+ as well as of isolated Fo reconstituted with F1 or F1-gamma subunit results in direct disulfide cross-linking between cysteine 197 in the carboxy-terminal region of the F0I-PVP(b) subunit and cysteine 91 at the carboxyl end of a small alpha-helix of subunit F1-gamma, both located in the stalk. The F0I-PVP(b) and F1-gamma cross-linking cause dramatic enhancement of oligomycin-sensitive decay of Delta mu H+. In ESMP and MgATP particles the cross-linking is accompanied by decoupling of respiratory ATP synthesis. These effects are consistent with the view that F0I-PVP(b) and F1-gamma are components of the stator and rotor of the proposed rotary motor, respectively. The fact that the carboxy-terminal region of F0I-PVP(b) and the short alpha-helix of F1-gamma can form a direct disulfide bridge shows that these two protein domains are, at least in the resting state of the enzyme, in direct contact. In isolated F0, diamide also induces cross-linking of OSCP with another subunit of F0, but this has no significant effect on proton conduction. When ESMP are treated with diamide in the presence of Delta mu H+ generated by respiration, neither cross-linking between F0I-PVP(b) and F1-gamma subunits nor the associated effects on proton conduction and ATP synthesis is observed. Cross-linking is restored in respiring ESMP by Delta mu H+ collapsing agents as well as by DCCD or oligomycin. These observations indicate that the torque generated by Delta mu H+ decay through Fo induces a relative motion and/or a separation of the F0I-PVP(b) subunit and F1-gamma which places the single cysteine residues, present in each of the two subunits, at a distance at which they cannot be engaged in disulfide bridging.
在不存在呼吸性ΔμH⁺的情况下，用二酰胺处理“内翻”亚线粒体颗粒（ESMP）中的F0F1 - ATP合酶，以及用F1或F1 - γ亚基重组的分离的F0，会导致F0I - PVP(b)亚基羧基末端区域的半胱氨酸197与F1 - γ亚基小α - 螺旋羧基端的半胱氨酸91之间直接形成二硫键交联，这两个半胱氨酸均位于柄部。F0I - PVP(b)与F1 - γ的交联导致ΔμH⁺对寡霉素敏感的衰减显著增强。在ESMP和MgATP颗粒中，交联伴随着呼吸性ATP合成的解偶联。这些效应与以下观点一致，即F0I - PVP(b)和F1 - γ分别是所提出的旋转马达定子和转子的组成部分。F0I - PVP(b)的羧基末端区域和F1 - γ的短α - 螺旋能够形成直接二硫键桥这一事实表明，这两个蛋白质结构域至少在酶的静止状态下是直接接触的。在分离的F0中，二酰胺也会诱导寡霉素敏感性蛋白（OSCP）与F0的另一个亚基交联，但这对质子传导没有显著影响。当在呼吸产生的ΔμH⁺存在下用二酰胺处理ESMP时，未观察到F0I - PVP(b)和F1 - γ亚基之间的交联以及对质子传导和ATP合成的相关影响。通过ΔμH⁺塌陷剂以及二环己基碳二亚胺（DCCD）或寡霉素，在呼吸的ESMP中交联得以恢复。这些观察结果表明，通过F0的ΔμH⁺衰减产生的扭矩会诱导F0I - PVP(b)亚基和F1 - γ发生相对运动和/或分离，从而使两个亚基中各自存在的单个半胱氨酸残基处于无法形成二硫键桥的距离。
