Fadda Elisa, Chakrabarti Nilmadhab, Pomès Régis
Structural Biology and Biochemistry, The Hospital for Sick Children, and Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada.
J Phys Chem B. 2005 Dec 1;109(47):22629-40. doi: 10.1021/jp052734+.
Cytochrome c oxidase (CcO) is a crucial enzyme in the respiratory chain. Its function is to couple the reduction of molecular oxygen, which takes place in the Fea3-CuB binuclear center, to proton translocation across the mitochondrial membrane. Although several high-resolution structures of the enzyme are known, the molecular basis of proton pumping activation and its mechanism remain to be elucidated. We examine a recently proposed scheme (J. Am. Chem. Soc. 2004, 126, 1858; FEBS Lett. 2004, 566, 126) that involves the deprotonation of the CuB-bound imidazole ring of a histidine (H291 in mammalian CcO) as a key element in the proton pumping mechanism. The central feature of that proposed mechanism is that the pKa values of the imidazole vary significantly depending on the redox state of the metals in the binuclear center. We use density functional theory in combination with continuum electrostatics to calculate the pKa values, successively in bulk water and within the protein, of the Cu-bound imidazole in various Cu- and Cu-Fe complexes. From pKas in bulk water, we derived a value of -266.34 kcal.mol(-1) for the proton solvation free energy (Delta). This estimate is in close agreement with the experimental value of -264.61 kcal.mol(-1) (J. Am. Chem. Soc. 2001, 123, 7314), which reinforces the conclusion that Delta is more negative than previous values used for pKa calculations. Our approach, on the basis of the study of increasingly more detailed models of the CcO binuclear center at different stages of the catalysis, allows us to examine successively the effect of each of the two metals' redox states and of solvation on the acidity of imidazole, whose pKa is approximately 14 in bulk water. This analysis leads to the following conclusions: first, the effect of Cu ligation on the imidazole acidity is negligible regardless of the redox state of the metal. Second, results obtained for Cu-Fe complexes in bulk water indicate that Cu-bound imidazole pKa values lie within the range of 14.8-16.6 throughout binuclear redox states corresponding to the catalytic cycle, demonstrating that the effect of the Fe oxidation states is also negligible. Finally, the low-dielectric CcO proteic environment shifts the acid-base equilibrium toward a neutral imidazole, further increasing the corresponding pKa values. These results are inconsistent with the proposed role of the Cu-bound histidine as a key element in the pumping mechanism. Limitations of continuum solvation models in pKa calculations are discussed.
细胞色素c氧化酶（CcO）是呼吸链中的一种关键酶。其功能是将在Fea3-CuB双核中心发生的分子氧还原与质子跨线粒体膜转运偶联起来。尽管该酶的几种高分辨率结构已为人所知，但质子泵浦激活的分子基础及其机制仍有待阐明。我们研究了最近提出的一种机制（《美国化学会志》2004年，126卷，1858页；《欧洲生物化学学会联合会快报》2004年，566卷，126页），该机制涉及组氨酸（哺乳动物CcO中的H291）与CuB结合的咪唑环的去质子化，这是质子泵浦机制中的关键要素。该机制的核心特征是咪唑的pKa值会根据双核中心中金属的氧化还原状态而显著变化。我们结合密度泛函理论和连续介质静电学，依次计算了各种Cu和Cu-Fe配合物中与Cu结合的咪唑在体相水中以及蛋白质内部的pKa值。根据体相水中的pKa值，我们得出质子溶剂化自由能（Δ）为-266.34 kcal·mol⁻¹。这一估计值与-264.61 kcal·mol⁻¹的实验值（《美国化学会志》2001年，123卷，7314页）非常吻合，这进一步强化了Δ比之前用于pKa计算的值更负的结论。我们的方法基于对催化不同阶段越来越详细的CcO双核中心模型的研究，使我们能够依次研究两种金属的氧化还原状态以及溶剂化对咪唑酸度的影响，咪唑在体相水中的pKa约为14。该分析得出以下结论：第一，无论金属的氧化还原状态如何，Cu配位对咪唑酸度的影响都可以忽略不计。第二，在体相水中对Cu-Fe配合物的研究结果表明，在对应催化循环的整个双核氧化还原状态下，与Cu结合的咪唑pKa值在14.8 - 16.6范围内，这表明Fe氧化态的影响也可以忽略不计。最后，低介电常数的CcO蛋白质环境使酸碱平衡向中性咪唑方向移动，进一步提高了相应的pKa值。这些结果与所提出的与Cu结合的组氨酸作为泵浦机制关键要素的作用不一致。文中还讨论了连续介质溶剂化模型在pKa计算中的局限性。
