Ma John K, Wang Yongting, Carrell Christopher J, Mathews F Scott, Davidson Victor L
Department of Biochemistry, The University of Mississippi Medical Center, Jackson, Mississippi 39216-4505, USA.
Biochemistry. 2007 Oct 2;46(39):11137-46. doi: 10.1021/bi7012307. Epub 2007 Sep 8.
Amicyanin is a type 1 copper protein that is the natural electron acceptor for the quinoprotein methylamine dehydrogenase (MADH). A P52G amicyanin mutation increased the Kd for complex formation and caused the normally true electron transfer (ET) reaction from O-quinol MADH to amicyanin to become a gated ET reaction (Ma, J. K., Carrell, C. J., Mathews, F. S., and Davidson, V. L. (2006) Biochemistry 45, 8284-8293). One consequence of the P52G mutation was to reposition the side chain of Met51, which is present at the MADH-amicyanin interface. To examine the precise role of Met51 in this interprotein ET reaction, Met51 was converted to Ala, Lys, and Leu. The Kd for complex formation of M51A amicyanin was unchanged but the experimentally determined electronic coupling increased from 12 cm-1 to 142 cm-1, and the reorganization energy increased from 2.3 to 3.1 eV. The rate and salt dependence of the proton transfer-gated ET reaction from N-quinol MADH to amicyanin is also changed by the M51A mutation. These changes in ET parameters and rates for the reactions with M51A amicyanin were similar to those caused by the P52G mutation and indicated that the ET reaction had become gated by a similar process, most likely a conformational rearrangement of the protein ET complex. The results of the M51K and M51L mutations also have consequences on the kinetic mechanism of regulation of the interprotein ET with effects that are intermediate between what is observed for the reaction of the native amicyanin and M51A amicyanin. These data indicate that the loss of the interactions involving Pro52 were primarily responsible for the change in Kd for P52G amicyanin, while the interactions involving the Met51 side chain are entirely responsible for the change in ET parameters and conversion of the true ET reaction of native amicyanin into a conformationally gated ET reaction.
氨腈蛋白是一种1型铜蛋白，是喹蛋白甲胺脱氢酶（MADH）的天然电子受体。P52G氨腈蛋白突变增加了复合物形成的解离常数（Kd），并导致从O - 喹醇MADH到氨腈蛋白的正常真实电子转移（ET）反应变成门控ET反应（Ma, J. K., Carrell, C. J., Mathews, F. S., and Davidson, V. L. (2006) Biochemistry 45, 8284 - 8293）。P52G突变的一个后果是重新定位了位于MADH - 氨腈蛋白界面的Met51的侧链。为了研究Met51在这种蛋白间ET反应中的精确作用，将Met51分别替换为Ala、Lys和Leu。M51A氨腈蛋白复合物形成的Kd没有变化，但实验测定的电子耦合从12 cm-1增加到142 cm-1，重组能从2.3 eV增加到3.1 eV。M5lA突变也改变了从N - 喹醇MADH到氨腈蛋白的质子转移门控ET反应的速率和盐依赖性。与M51A氨腈蛋白反应的这些ET参数和速率变化与P52G突变引起的变化相似，表明ET反应通过类似的过程变成了门控反应，最有可能是蛋白ET复合物的构象重排。M51K和M51L突变的结果也对蛋白间ET调节的动力学机制产生影响，其作用介于天然氨腈蛋白和M51A氨腈蛋白反应所观察到的情况之间。这些数据表明，涉及Pro52的相互作用的丧失主要是P52G氨腈蛋白Kd变化的原因，而涉及Met51侧链的相互作用完全是ET参数变化以及天然氨腈蛋白的真实ET反应转变为构象门控ET反应的原因。
