Density Functional Theory Approach to Biochemical Reactions

从B3LYP DFT计算出发，讨论了细胞色素P450的铁氧化合物I将樟脑转化为5-外羟基樟脑的机理和能量方面。这个反应以两步的方式发生，沿着氧反弹机制的思路。计算出樟脑C5原子h原子抽离的第一跃迁态活化能大于20 kcal/mol。h原子的提取是氢化反应的速率决定步骤，导致反应中间体包含碳自由基和铁羟基。连接反应中间体和产物醇络合物的反弹步骤的第二个过渡态比双重态和四重态势能表面上的h原子抽象的过渡态低几千卡/摩尔。这一高能特性使得在两种自旋状态下几乎没有障碍的重组，与实验观察到的高强度反应相一致。还考虑和计算了樟脑羟基化催化机制的整体能量分布，特别是关于为什么在生理条件下可以获得h原子抽象的高活化能。根据涉及Thr252、Asp251和两种溶剂水分子的质子源模型，研究了过氧化铁类转化为化合物I的能量学。在这个双氧活化过程中释放了超过50千卡/摩尔的能量。通过理论计算，研究了多巴胺β-单加氧酶（DBM）的铜-超氧、-氢过氧和-氧基团对多巴胺的羟基化作用，以确定其反应中的活性基团，并揭示其周围氨基酸残基在底物结合中的关键功能。以序列同源性高达30%的肽基甘氨酸α-羟化单加氧酶（PHM）晶体结构为模板，通过同源性建模，构建了大鼠DBM的三维模型。在构建的三维模型中，区域1中的CuA位点由三个组氨酸残基His265、His266和His336协调，而区域2中的CuB位点由两个组氨酸残基His415和His417以及一个蛋氨酸残基Met490协调。三个Glu268， Glu369和Tyr494残基被认为在DBM活性位点的底物结合中发挥重要作用，从而实现立体定向氢原子的提取。通过量子力学/分子力学（QM/MM）计算，确定了铜-超氧、-氢过氧和-氧在约4700个原子的全酶模型中的结构和催化机理。在全酶模型的计算中，铜-氧的反应性是显著的。少

Mechanistic and energetic aspects for the conversion of camphor to 5-exo-hydroxycamphor by the compound I iron-oxo species of cytochrome P450 are discussed from B3LYP DFT calculations. This reaction occurs in a two-step manner along the lines that the oxygen rebound mechanism suggests. The activation energy for the first transition state of the H-atom abstraction at the C5 atom of camphor is computed to be more the 20 kcal/mol. This H-atom abstraction is the rate-determining step in this hydroxylation reaction, leading to a reaction intermediate that involves a carbon radical species and the iron hydroxo species. The second transition state of the rebound step that connects the reaction intermediate and the product alcohol complex lies a few kcal/mol below that for the H-atom abstraction on the doublet and quartet potential energy surfaces. This energetic feature allows the virtually barrierless recombination in both spin states, being consistent with experimentally observed high stere … More oselectivity and brief lifetimes of the reaction intermediate. The overall energetic profile of the catalytic mechanism of camphor hydroxylation particularly with respect to why the high activation energy for the H-atom abstraction is accessible under physiological conditions is also considered and calculated. According to a proton source model involving Thr252,Asp251, and two solvent water molecules, the energetics for the conversion of the iron-peroxo species to compound I is studied. A significant energy over 50 kcal/mol is released in the course of this dioxygen activation process. Dopamine hydroxylation by the copper-superoxo, -hydroperoxo, and -oxo species of dopamine β-monooxygenase(DBM) is investigated from theoretical calculations to identify the active species in its reaction and to reveal the key functions of the surrounding amino acid residues in substrate binding. A 3D model of rat DBM is constructed by homology modeling using the crystal structure of peptidylglycine α-hydroxylating monooxygenase(PHM) with a high sequence identity of 30% as a template. In the constructed 3D model, the CuA site in domain 1 is coordinated by three histidine residues, His265, His266, and His336, while the CuB site in domain 2 is coordinated by two histidine residues, His415 and His417, and by a methionine residue Met490. The three Glu268, Glu369, and Tyr494 residues are suggested to play an important role in the substrate binding at the active site of DBM to enable the stereospecific hydrogen-atom abstraction. Quantum mechanical/molecular mechanical (QM/MM) calculations are performed to determine the structure and catalytic mechanism of the copper-superoxo, -hydroperoxo, and -oxo species in the whole-enzyme model with about 4,700 atoms. The reactivity of the copper-oxo species is significant from calculations of the whole-enzyme model. Less