Christensen H E, Ulstrup J, Sykes A G
Department of Chemistry, The University, Newcastle upon Tyne, U.K.
Biochim Biophys Acta. 1990 May 31;1039(1):94-102. doi: 10.1016/0167-4838(90)90231-4.
Plastocyanin (PCu) from spinach leaves has been singly NO2-modified, purified by FPLC, and the position of modification at Tyr83 confirmed by trypsin digestion and amino-acid sequencing. Electron-transfer reactions of native and NO2-modified PCu with the inorganic redox partners [Fe(CN)6]3- and [Co(phen)3]3+, as oxidants for PCu(I), and [Fe(CN)6]4- and [Co(phen)3]2+ as reductants for PCu(II), have been studied as a function of pH. The acid dissociation constant for the phenolic group on NO2-Tyr83 PCu is 8.78 (average) for reduced, and 8.10 for oxidised protein, as compared to values greater than 10 for native protein. At I = 0.10 M (NaCl) NO2-modification brings about a 20 mV increase in reduction potential at pH less than 7 and deprotonation of the phenolic group a 20-25 mV decrease, both transmitted to and effective at the active site. Deprotonation brings about a 48% increase in rate for [Fe(CN)6]3- and a 47% decrease for [Fe(CN)6]4- in accordance with these changes. In the case of [Co(phen)3]3+, which reacts substantially at the remote site in the vicinity of Tyr83, the influence of deprotonation on the active site is supplemented by the negative charge of the phenolate, and a total increase of 131% is observed. These results can be understood on the basis of the electron-transfer theory, and add support to the belief that electron transfer kinetics of negatively and positively charged reactants are dominated by different sites on PCu for electron transfer, namely adjacent (close to His87) and remote (close to Tyr83), respectively.
菠菜叶中的质体蓝素（PCu）已被单独进行NO₂修饰，通过快速蛋白质液相色谱（FPLC）纯化，并通过胰蛋白酶消化和氨基酸测序确定了Tyr83的修饰位置。研究了天然和NO₂修饰的PCu与无机氧化还原伙伴[Fe(CN)₆]³⁻和[Co(phen)₃]³⁺（作为PCu(I)的氧化剂）以及[Fe(CN)₆]⁴⁻和[Co(phen)₃]²⁺（作为PCu(II)的还原剂）之间的电子转移反应，作为pH的函数。与天然蛋白质大于10的值相比，NO₂-Tyr83 PCu上酚基团的酸解离常数在还原态时平均为8.78，氧化态时为8.10。在I = 0.10 M（NaCl）时，NO₂修饰在pH小于7时使还原电位增加20 mV，酚基团去质子化使还原电位降低20 - 25 mV，这两种变化都传递到活性位点并在活性位点起作用。去质子化导致[Fe(CN)₆]³⁻的反应速率增加48%，[Fe(CN)₆]⁴⁻的反应速率降低47%，与这些变化一致。对于在Tyr83附近的远程位点发生显著反应的[Co(phen)₃]³⁺，去质子化对活性位点的影响由酚盐的负电荷补充，观察到总增加量为131%。这些结果可以基于电子转移理论来理解，并支持了这样一种观点，即带负电荷和带正电荷反应物的电子转移动力学分别由PCu上不同的电子转移位点主导，即相邻位点（靠近His87）和远程位点（靠近Tyr83）。
