Spectroscopic insights into axial ligation and active-site H-bonding in substrate-bound human heme oxygenase-2
Spectroscopic insights into axial ligation and active-site H-bonding in substrate-bound human heme oxygenase-2
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DOI：
10.1007/s00775-010-0672-8
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发表时间：
2010-09-01
影响因子：
3
通讯作者：
Brunold, Thomas C.
中科院分区：
文献类型：
--
作者：
Gardner, Jessica D.;Yi, Li;Brunold, Thomas C.
血红素加氧酶(HOS)是一种单加氧酶，它催化血红素降解的第一步，将血红素转化为胆绿素，同时从卟啉大循环中释放Fe(II)和CO。存在两种不同的血红素加氧酶亚型，HO-1和HO-2，包括HO-1中完全缺乏半胱氨酸残基，以及HO-2中存在三个半胱氨酸残基作为血红素调节基序(HRMS)的一部分。其他血红素蛋白中的HRMS被认为直接与血红素结合，或以其他方式调节蛋白质的稳定性或活性；然而，目前尚不清楚HRMS如何在HO-2功能中发挥这些作用。为了更好地了解这种重要酶的性质并阐明其HRMS的可能作用，人们制备了对HRMS具有明显变化的各种形式的HO-2。在这项研究中，将具有硫醇形式的Cys265的变体与具有氧化形式(部分二硫键或以亚硫酸盐部分存在)形式的残基的变体进行比较。这些HO-2变体的吸收光谱和磁圆二色谱数据清楚地表明，当Cys265被还原时，形成了一种新的低自旋Fe(III)血红素物种，其特征是硫代连接。此外，在不同温度下收集的吸收、磁圆二色谱和共振拉曼数据揭示了血红素-HO-2络合物中铁自旋态的有趣的温度依赖关系。这些发现与HO-2活性部位内血红素远端存在氢键网络是一致的，与在HO-1中观察到的有潜在的显着差异。
Heme oxygenases (HOs) are monooxygenases that catalyze the first step in heme degradation, converting heme to biliverdin with concomitant release of Fe(II) and CO from the porphyrin macrocycle. Two heme oxygenase isoforms, HO-1 and HO-2, exist that differ in several ways, including a complete lack of Cys residues in HO-1 and the presence of three Cys residues as part of heme-regulatory motifs (HRMs) in HO-2. HRMs in other heme proteins are thought to directly bind heme, or to otherwise regulate protein stability or activity; however, it is not currently known how the HRMs exert these effects on HO-2 function. To better understand the properties of this vital enzyme and to elucidate possible roles of its HRMs, various forms of HO-2 possessing distinct alterations to the HRMs were prepared. In this study, variants with Cys265 in a thiol form are compared with those with this residue in an oxidized (part of a disulfide bond or existing as a sulfenate moiety) form. Absorption and magnetic circular dichroism spectroscopic data of these HO-2 variants clearly demonstrate that a new low-spin Fe(III) heme species characteristic of thiolate ligation is formed when Cys265 is reduced. Additionally, absorption, magnetic circular dichroism, and resonance Raman data collected at different temperatures reveal an intriguing temperature dependence of the iron spin state in the heme-HO-2 complex. These findings are consistent with the presence of a hydrogen-bonding network at the heme's distal side within the active site of HO-2 with potentially significant differences from that observed in HO-1.