Lukoyanov Dmitriy, Berry Steven M, Lu Yi, Antholine William E, Scholes Charles P
Department of Chemistry and Center for Biological Macromolecules, University at Albany, SUNY, Albany, New York 12222, USA.
Biophys J. 2002 May;82(5):2758-66. doi: 10.1016/S0006-3495(02)75616-6.
The binuclear Cu(A) site engineered into Pseudomonas aeruginosa azurin has provided a Cu(A)-azurin with a well-defined crystal structure and a CuSSCu core having two equatorial histidine ligands, His120 and His46. The mutations His120Asn and His120Gly were made at the equatorial His120 ligand to understand the histidine-related modulation to Cu(A), notably to the valence delocalization over the CuSSCu core. For these His120 mutants Q-band electron nuclear double resonance (ENDOR) and multifrequency electron paramagnetic resonance (EPR) (X, C, and S-band), all carried out under comparable cryogenic conditions, have provided markedly different electronic measures of the mutation-induced change. Q-band ENDOR of cysteine C(beta) protons, of weakly dipolar-coupled protons, and of the remaining His46 nitrogen ligand provided hyperfine couplings that were like those of other binuclear mixed-valence Cu(A) systems and were essentially unperturbed by the mutation at His120. The ENDOR findings imply that the Cu(A) core electronic structure remains unchanged by the His120 mutation. On the other hand, multifrequency EPR indicated that the H120N and H120G mutations had changed the EPR hyperfine signature from a 7-line to a 4-line pattern, consistent with trapped-valence, Type 1 mononuclear copper. The multifrequency EPR data imply that the electron spin had become localized on one copper by the His120 mutation. To reconcile the EPR and ENDOR findings for the His120 mutants requires that either: if valence localization to one copper has occurred, the spin density on the cysteine sulfurs and the remaining histidine (His46) must remain as it was for a delocalized binuclear Cu(A) center, or if valence delocalization persists, the hyperfine coupling for one copper must markedly diminish while the overall spin distribution on the CuSSCu core is preserved.
工程改造到铜绿假单胞菌天青蛋白中的双核铜（A）位点为铜（A）-天青蛋白提供了明确的晶体结构以及具有两个赤道面组氨酸配体（His120和His46）的CuSSCu核心。在赤道面His120配体处进行了His120Asn和His120Gly突变，以了解组氨酸对铜（A）的相关调节作用，特别是对CuSSCu核心上价态离域的影响。对于这些His120突变体，在可比的低温条件下进行了Q波段电子核双共振（ENDOR）和多频电子顺磁共振（EPR）（X、C和S波段），结果显示突变诱导变化的电子测量结果明显不同。半胱氨酸C（β）质子、弱偶极耦合质子以及其余His46氮配体的Q波段ENDOR提供了超精细耦合，这些耦合与其他双核混合价铜（A）系统的耦合相似，并且基本上不受His120处突变的影响。ENDOR结果表明，His120突变并未改变铜（A）核心的电子结构。另一方面，多频EPR表明，H120N和H120G突变使EPR超精细特征从7线模式变为4线模式，这与捕获价态的1型单核铜一致。多频EPR数据表明，His120突变使电子自旋定域在一个铜原子上。要协调His120突变体的EPR和ENDOR结果，需要满足以下两种情况之一：如果发生了价态定域到一个铜原子上的情况，那么半胱氨酸硫原子和其余组氨酸（His46）上的自旋密度必须保持为离域双核铜（A）中心时的状态；或者，如果价态离域仍然存在，那么一个铜原子的超精细耦合必须显著减弱，同时保持CuSSCu核心上的整体自旋分布。
