Kinetic isotope effects on the rate-limiting step of heme oxygenase catalysis indicate concerted proton transfer/heme hydroxylation
Kinetic isotope effects on the rate-limiting step of heme oxygenase catalysis indicate concerted proton transfer/heme hydroxylation
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DOI：
10.1021/ja038923s
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发表时间：
2003-12-31
影响因子：
15
通讯作者：
Hoffman, BM
中科院分区：
文献类型：
--
作者：
Davydov, R;Matsui, T;Hoffman, BM
血红素加氧酶(HO)通过血红素作为氧活化辅基和底物参与的过程，催化依赖于O2和NADPH/细胞色素P450的还原酶将血红素转化为胆绿素、游离铁离子和一氧化碳。我们早些时候证实了HO催化的第一步是单氧化，其中一个电子和两个质子与HO氧铁血红素加成生成铁-α-中羟基血红素(H)。冷冻还原/电子顺磁共振和Endor测量进一步表明，氢过氧铁-HO在一个动力学步骤中直接转化为HH，而不形成化合物I。我们在这里报告了该限速步骤的细节。人氧-Ho的单电子77K低温还原和200K的热处理产生了一个结构松弛的氢过氧铁-Ho物种，命名为R。本文报道了低温还原/退火法直接测定限速R-→H转化的溶剂和次级动力学同位素效应(Kie)的实验，该实验使用由中间氢血红素制备的酶和在H2O/D2O缓冲液中测定溶剂Kie(solv-Kie)和与转化相关的次级Kie(Secc-Kie)。这种方法的独特之处在于，通过监测限速步骤测量的KIE不容易被其他过程的KIE所掩盖，并且这些结果首次直接测量了由氧活化血红素酶中的动力学活性反应中间产物形成的KIE。观察到两者的solv-Kie(298)=1.8和Sec-Kie(298)=0.8(相反)表明，hby HO的形成的限速步骤是一个协调的过程： 质子通过远端口袋H-键网络转移到氢过氧铁-血红素，很可能来自作为一般酸催化剂的羧基，与末端氢过氧氧原子和α−中间碳之间的键形成同步发生，形成四面体羟化血红素中间体。随后的重排和H2O的损失就会产生。
Heme oxygenase (HO) catalyzes the O2and NADPH/cytochrome P450 reductase-dependent conversion of heme to biliverdin, free iron ion, and CO through a process in which the heme participates as both dioxygen-activating prosthetic group and substrate. We earlier confirmed that the first step of HO catalysis is a monooxygenation in which the addition of one electron and two protons to the HO oxy-ferroheme produces ferric-α-meso-hydroxyheme (h). Cryoreduction/EPR and ENDOR measurements further showed that hydroperoxo-ferri-HO converts directly tohin a single kinetic step without formation of a Compound I. We here report details of that rate-limiting step. One-electron 77 K cryoreduction of human oxy-HO and annealing at 200 K generates a structurally relaxed hydroperoxo-ferri-HO species, denotedR. We here report the cryoreduction/annealing experiments thatdirectlymeasure solvent and secondary kinetic isotope effects (KIEs) of the rate-limitingR→hconversion, using enzyme prepared withmeso-deuterated heme and in H2O/D2O buffers to measure the solvent KIE (solv-KIE), and the secondary KIE (sec-KIE) associated with the conversion. This approach is unique in that KIEs measured by monitoring the rate-limiting step are not susceptible to masking by KIEs of other processes, and these results represent the first direct measurement of the KIEs of product formation by a kinetically competent reaction intermediate inanydioxygen-activating heme enzyme.The observation ofbothsolv-KIE(298) = 1.8 and sec-KIE(298) = 0.8 (inverse) indicates that the rate-limiting step for formation ofhby HO is a concerted process: proton transfer to the hydroperoxo-ferri-heme through the distal-pocket H-bond network, likely from a carboxyl group acting as a general acid catalyst, occurring in synchrony with bond formation between the terminal hydroperoxo-oxygen atom and the α−mesocarbon to form a tetrahedral hydroxylated-heme intermediate. Subsequent rearrangement and loss of H2O then generatesh.