Catalytic mechanism of heme oxygenase through EPR and ENDOR of cryoreduced oxy-heme oxygenase and its Asp 140 mutants
Catalytic mechanism of heme oxygenase through EPR and ENDOR of cryoreduced oxy-heme oxygenase and its Asp 140 mutants
复制标题
DOI：
10.1021/ja0122391
复制
发表时间：
2002-02-27
影响因子：
15
通讯作者：
Hoffman, BM
中科院分区：
文献类型：
--
作者：
Davydov, R;Kofman, V;Hoffman, BM
血红素加氧酶（HO）催化O-2和nadph -细胞色素P450还原酶依赖的血红素转化为胆绿素、铁和一氧化碳，在这个过程中血红素既作为假基又作为底物参与。在本研究中，我们生成了HO催化的第一个单氧化步骤，血红素转化为α -中羟基血红素的详细反应周期。我们使用EPR(同时使用O-16（2)和O-17(2)）和H-1， N-14 ENDOR光谱来表征野生型氧- ho和D140A， F突变体经77 K辐射冷冻还原和随后退火后产生的中间体。氧- ho的单电子低温还原得到一个g张量为g =[2,37, 2.187, 1.924]的氢-过氧- ho。将该物质退火至200 K时，伴随着光谱变化，其中包括新的H-1 ENDOR信号的出现，反映了活性位点的重排。在214 K下的动力学测量表明，退火的氢过氧铁- ho (R)在一级反应中生成了铁- α -中羟基血红素产物。残基D140的丙氨酸和苯丙氨酸突变破坏了HO远端口袋内的h键网络，从而阻止了产物的形成。而氢过氧铁- ho （D140A）则发生O-O键的异裂，最终生成不形成产物的epr沉默化合物ii类物质。这些结果与先前的建议一致，确定了氢过氧铁- ho确实是反应物质，通过氢过氧部分在血红素α -碳上的远端OH的攻击直接形成α -中位羟基血红素产物。
Heme oxygenase (HO) catalyzes the O-2- and NADPH-cytochrome P450 reductase-dependent conversion of heme to biliverdin, Fe, and CO through a process in which the heme participates both as a prosthetic group and as a substrate. In the present study, we have generated a detailed reaction cycle for the first monooxygenation step of HO catalysis, conversion of the heme to alpha-meso-hydroxyheme. We employed EPR (using both O-16(2) and O-17(2)) and H-1, N-14 ENDOR spectroscopies to characterize the intermediates generated by 77 K radiolytic cryoreduction and subsequent annealing of wild-type oxy-HO and D140A, F mutants. One-electron cryoreduction of oxy-HO yields a hydroperoxoferri-HO with g-tensor, g = [2,37, 2.187, 1.924]. Annealing of this species to 200 K is accompanied by spectroscopic changes that include the appearance of a new H-1 ENDOR signal, reflecting rearrangements in the active site. Kinetic measurements at 214 K reveal that the annealed hydroperoxoferri-HO species, denoted R, generates the ferri-alpha-meso-hydroxyheme product in a first-order reaction. Disruption of the H-bonding network within the distal pocket of HO by the alanine and phenylalanine mutations of residue D140 prevents product formation. The hydroperoxoferri-HO (D140A) instead undergoes heterolytic cleavage of the O-O bond, ultimately yielding an EPR-silent compound II-like species that does not form product. These results, which agree with earlier suggestions, establish that hydroperoxoferri-HO is indeed the reactive species, directly forming the alpha-meso-hydroxyheme product by attack of the distal OH of the hydroperoxo moiety at the heme alpha-carbon.