Department of Biochemistry, Faculty of Science, P. J. Šafárik University in Košice, Košice, 04154, Slovakia.
Centre for Interdisciplinary Biosciences, P. J. Šafárik University in Košice, Košice, 04154, Slovakia.
Biochemistry (Mosc). 2021 Jan;86(1):59-73. doi: 10.1134/S0006297921010065.
Specific effects of anions on the structure, thermal stability, and peroxidase activity of native (state III) and alkaline (state IV) cytochrome c (cyt c) have been studied by the UV-VIS absorbance spectroscopy, intrinsic tryptophan fluorescence, and circular dichroism. Thermal and isothermal denaturation monitored by the tryptophan fluorescence and circular dichroism, respectively, implied lower stability of cyt c state IV in comparison with the state III. The pK value of alkaline isomerization of cyt c depended on the present salts, i.e., kosmotropic anions increased and chaotropic anions decreased pK (Hofmeister effect on protein stability). The peroxidase activity of cyt c in the state III, measured by oxidation of guaiacol, showed clear dependence on the salt position in the Hofmeister series, while cyt c in the alkaline state lacked the peroxidase activity regardless of the type of anions present in the solution. The alkaline isomerization of cyt c in the presence of 8 M urea, measured by Trp59 fluorescence, implied an existence of a high-affinity non-native ligand for the heme iron even in a partially denatured protein conformation. The conformation of the cyt c alkaline state in 8 M urea was considerably modulated by the specific effect of anions. Based on the Trp59 fluorescence quenching upon titration to alkaline pH in 8 M urea and molecular dynamics simulation, we hypothesize that the Lys79 conformer is most likely the predominant alkaline conformer of cyt c. The high affinity of the sixth ligand for the heme iron is likely a reason of the lack of peroxidase activity of cyt c in the alkaline state.
本研究采用紫外可见吸收光谱、内源色氨酸荧光和圆二色性等方法，研究了阴离子对天然（III 态）和碱性（IV 态）细胞色素 c（cyt c）结构、热稳定性和过氧化物酶活性的特定影响。分别通过色氨酸荧光和圆二色性监测的热变性和等温变性表明，与 III 态相比，IV 态 cyt c 的稳定性较低。cyt c 的碱性异构化的 pK 值取决于存在的盐，即，亲溶质阴离子增加而疏溶质阴离子降低 pK（对蛋白质稳定性的 Hofmeister 效应）。通过氧化愈创木酚测量的 III 态 cyt c 的过氧化物酶活性，显示出与 Hofmeister 系列中盐位置的明显依赖性，而碱性状态的 cyt c 无论溶液中存在哪种类型的阴离子，均缺乏过氧化物酶活性。用色氨酸 59 荧光测量的存在 8 M 脲时 cyt c 的碱性异构化，暗示即使在部分变性的蛋白质构象中，血红素铁也存在高亲和力的非天然配体。8 M 脲中 cyt c 碱性状态的构象受到阴离子特异性效应的显著调节。基于在 8 M 脲中碱性 pH 滴定时色氨酸 59 荧光猝灭和分子动力学模拟，我们假设 Lys79 构象最有可能是 cyt c 的主要碱性构象。第六配体对血红素铁的高亲和力可能是 cyt c 在碱性状态下缺乏过氧化物酶活性的原因。
