Banci L, Bertini I, Capannoli C, Del Conte R, Tien M
Department of Chemistry, Centro di Risononze Magnetiche, University of Florence, Italy.
Biochemistry. 1999 Jul 27;38(30):9617-25. doi: 10.1021/bi9825697.
The mutants at position 242 of manganese peroxidase (MnP), where the native Asp has been substituted with a Ser or a Glu, have been shown to be active, and are here characterized by electronic, EPR, and NMR spectroscopies. We have also mutated another residue on the proximal side, Phe 190 to Val and Leu, yielding active mutants. When studied by the above-mentioned spectroscopies, the mutants at both positions 242 and 190 exhibit three pH-dependent transitions. In contrast to the transitions observed at low and high pH, the spectroscopic studies reveal that the transition at intermediate pH has pK(a) values up to 2 units lower for the mutants at D242E and -S and F190V than for the wild type. This process is due to the ionization of a group that affects the transition to the bis-histidine coordination at the iron. The observed changes in the pK(a) values are related to the altered affinity of the calcium-binding site in the distal pocket. Other variations are observed in the other two pK(a) values. Characterization of the cyanide derivatives indicates that the location and orientation of the distal and proximal His residues are essentially identical to that in the wild type. Our results indicate that mutations on the proximal side residues can affect changes in the distal side. In particular, deprotonation of a group, whose pK(a) is influenced by the nature of the residues in the proximal side, produces a movement of helix B, which in turn induces the coordination of the distal His and the loss of the distal calcium ion.
锰过氧化物酶（MnP）第242位的突变体，即天然天冬氨酸被丝氨酸或谷氨酸取代的突变体，已被证明具有活性，在此通过电子光谱、电子顺磁共振光谱和核磁共振光谱对其进行表征。我们还将近端的另一个残基苯丙氨酸190突变为缬氨酸和亮氨酸，得到了活性突变体。当通过上述光谱进行研究时，第242位和190位的突变体均表现出三种pH依赖性转变。与在低pH和高pH下观察到的转变不同，光谱研究表明，对于D242E和-S以及F190V突变体，中间pH下的转变的pK(a)值比野生型低多达2个单位。这个过程是由于一个基团的电离，该基团影响向铁的双组氨酸配位的转变。观察到的pK(a)值变化与远端口袋中钙结合位点亲和力的改变有关。在其他两个pK(a)值中也观察到了其他变化。氰化物衍生物的表征表明，远端和近端组氨酸残基的位置和取向与野生型基本相同。我们的结果表明，近端侧残基的突变可以影响远端侧的变化。特别是，一个pK(a)受近端侧残基性质影响的基团的去质子化会导致螺旋B的移动，进而诱导远端组氨酸的配位和远端钙离子的丢失。
