Christiansen J, Chan J M, Seefeldt L C, Dean D R
Department of Biochemistry, Fralin Biotechnology Center, Virginia Tech, Blacksburg 24061, USA.
J Inorg Biochem. 2000 Jul 1;80(3-4):195-204. doi: 10.1016/s0162-0134(00)00083-0.
Site-directed mutagenesis and gene-replacement techniques were used to substitute alanine for the MoFe protein alpha- and beta-subunit phenylalanine-125 residues both separately and in combination. These residues are located on the surface of the MoFe protein near the pseudosymmetric axis of symmetry between the alpha- and beta-subunits. Altered MoFe proteins that contain an alanine substitution at only one of the respective positions exhibit proton reduction activities of about 25-50% when compared to that of the wild-type protein. The lower level of proton reduction also corresponds with decreases in the rates of MgATP hydrolysis. The MoFe protein which contains alanine substitutions in both the alpha- and beta- subunits did not exhibit any proton reduction activity or MgATP hydrolysis. Stopped flow spectrophotometry of the singly substituted MoFe proteins indicate primary electron transfer rate constants approximately an order of magnitude slower than what is observed for wild-type MoFe protein, while no primary electron transfer is observed for the doubly substituted MoFe protein. The doubly substituted MoFe protein is able to interact with the Fe protein as shown by chemical crosslinking experiments. However, this protein does not form a tight complex with the Fe protein when treated with MgADP-AlF4- or when using the altered 127delta Fe protein. Stopped flow spectrophotometry was also used to quantitate the first-order dissociation rate constants for the two component proteins. These results suggest that the 125Phe residues are involved in an early event(s) that occurs upon component protein docking and could be involved in eliciting MgATP hydrolysis.
采用定点诱变和基因置换技术，分别或联合将丙氨酸替代钼铁蛋白α亚基和β亚基的苯丙氨酸-125残基。这些残基位于钼铁蛋白表面，靠近α亚基和β亚基之间的假对称对称轴。与野生型蛋白相比，仅在各自位置之一含有丙氨酸替代的改变后的钼铁蛋白表现出约25%-50%的质子还原活性。较低水平的质子还原也与MgATP水解速率的降低相对应。在α亚基和β亚基中均含有丙氨酸替代的钼铁蛋白未表现出任何质子还原活性或MgATP水解活性。对单取代钼铁蛋白的停流分光光度法表明，其初级电子转移速率常数比野生型钼铁蛋白慢约一个数量级，而双取代钼铁蛋白未观察到初级电子转移。化学交联实验表明，双取代钼铁蛋白能够与铁蛋白相互作用。然而，当用MgADP-AlF4-处理或使用改变后的127δ铁蛋白时，该蛋白与铁蛋白不会形成紧密复合物。停流分光光度法也用于定量两种组分蛋白的一级解离速率常数。这些结果表明，125位苯丙氨酸残基参与了组分蛋白对接时发生的早期事件，并且可能参与引发MgATP水解。
