Aubert C, Giudici-Orticoni M T, Czjzek M, Haser R, Bruschi M, Dolla A
Unite de Bioenergetique et Ingenierie de Proteines, UPR 9036, CNRS, 13402 Marseille Cedex 20, France.
Biochemistry. 1998 Feb 24;37(8):2120-30. doi: 10.1021/bi971656g.
A combination of structural, kinetic, and interaction experiments has been used to study the role of a highly conserved aromatic residue, Tyr73, parallel to the sixth heme axial ligand of heme 4 in multiheme cytochrome c3 (Mr = 26 000), also called cytochrome cc3 or octaheme cytochrome, from Desulfovibrio desulfuricans Norway. This residue is expected to be involved in intermolecular electron transfer and protein-protein interaction, since heme 4 is described to be the interaction site between physiological partners. The kinetic experiments show that the Y73E replacement provokes no significant change in the electron-transfer reaction with the physiological partner, the [NiFeSe] hydrogenase, but that the protein-protein interaction between cytochrome c3 (Mr = 26 000) and hydrogenase is strongly affected by the mutation. The aromatic residue does not play a role in maintaining the axial heme ligand in a particular orientation, since the mutation did not affect the orientation of histidine 77, the sixth axial ligand of heme 4. The structural analysis by X-ray crystallography clearly shows that a rearrangement of the charged residues in the vicinity of the mutation site is responsible for the change in protein-protein interaction, which is of an electrostatic nature. Lys22 and Arg66, residues which are located at the interacting surface, are twisted toward the mutated position Glu73 in order to compensate for the negative charge and therefore are no longer accessible for the docking with a physiological partner. Tyr73 has instead a structural function and probably a role in maintaining the hydrophobic environment of the heme 4 cavity rather than a function in the intermolecular electron transfer with the physiological partners.
通过结构、动力学和相互作用实验相结合的方法，研究了高度保守的芳香族残基Tyr73的作用。该残基与来自挪威脱硫脱硫弧菌的多血红素细胞色素c3（Mr = 26000，也称为细胞色素cc3或八血红素细胞色素）中血红素4的第六个血红素轴向配体平行。由于血红素4被描述为生理伴侣之间的相互作用位点，预计该残基参与分子间电子转移和蛋白质 - 蛋白质相互作用。动力学实验表明，Y73E替换对与生理伴侣[NiFeSe]氢化酶的电子转移反应没有显著影响，但细胞色素c3（Mr = 26000）与氢化酶之间的蛋白质 - 蛋白质相互作用受到该突变的强烈影响。该芳香族残基在维持轴向血红素配体处于特定方向方面不起作用，但该突变并不影响血红素4的第六个轴向配体组氨酸77的方向。X射线晶体学的结构分析清楚地表明，突变位点附近带电残基的重排导致了蛋白质 - 蛋白质相互作用的变化，这种变化具有静电性质。位于相互作用表面的残基Lys22和Arg66向突变位置Glu73扭转，以补偿负电荷，因此不再可用于与生理伴侣对接。相反，Tyr73具有结构功能，可能在维持血红素4腔的疏水环境中起作用，而不是在与生理伴侣的分子间电子转移中起作用。
