Understanding the Selectivity of Oxygen Activation by Model Iron-Porphyrin Catalysts

项目摘要 含血红素蛋白是自然界中最丰富的金属蛋白之一。一个重要的子集 这些催化剂利用包括细胞色素c氧化酶（CcO）的分子氧（O2）进行反应， 细胞色素P450（CYP 450），其活化分子氧以氧化有机物 印刷受体. CO2和CCO的功能依赖于它们将O2还原为水的能力。在一个“非耦合” 在该过程中，一些当量的还原剂被浪费，并且活性氧物质（ROS）如H2 O2 发布众所周知，ROS会导致人体内的氧化应激和多种疾病， 对这种脱钩的理解是重要的。据推测，催化Fe-OOH 中间体是H2O和H2 O2形成之间的分叉点。该过氧化氢中间体是 在含血红素的酶中普遍存在，包括细胞色素c过氧化物酶、血红素加氧酶和 前列腺素H合酶在所有情况下，适当的质子传递对于O-O键断裂是必要的。 P450 cam的诱变研究表明质子和氢键的作用是重要的 在理解选择性，但有争议的保守残基如何防止解偶联。 此外，正则机制涉及一个质子添加到近端或远端氧原子 的Fe-OOH中间体，分别产生H2 O2或H2O，但没有直接证据表明这一点 化学计量我们的初步数据表明，所需的远端质子化可能涉及更高的 依赖于质子。因此，我们的目标是研究是什么决定了H2O形成之间的选择性 或从该关键Fe-OOH中间体释放H2 O2。 我们的工作在这个建议试图了解H2O和H2 O2形成的选择性血红素 利用简单的模型系统的酶。合成类似物的优势在于， 系统地改变和控制结构实体，在新的地方进入催化循环， 合成中间体。因此，我们建议研究铁卟啉活化O2的选择性 催化剂通过相同的Fe-OOH中间体进行。首先，我们将探讨各种反应 条件（酸的浓度、pKa和结构）影响催化氧还原中的选择性 反应（ORR）。其次，我们将研究具有不同氢键基序的各种催化剂，以更好地理解 活性位点上的残基如何影响选择性。此外，我们将探索非催化性 Fe-OOH中间体的反应性，以获得H2O和H2 O2相对速率的独立测量 在不同条件下形成。最终，我们的目标是了解反应条件和H- 键合网络影响模型系统中H2O对H2 O2的选择性。这种理解将提供洞察力 酶如何控制关键的Fe-过氧化氢中间体的反应性，以最大限度地减少ROS 在各种含血红素的活性部位形成。

PROJECT SUMMARY Heme-containing proteins are among the most abundant metalloproteins in nature. A significant subset of these catalysts perform reactions utilizing dioxygen (O2), including cytochrome c oxidase (CcO) which reduces dioxygen to water, and cytochrome P450 (CYP) which activates dioxygen in order to oxidize organic substrates. The functions of CYP and CcO rely on their abilities to reduce O2 to water. In an “uncoupled” process, some equivalents of reductant are wasted and reactive oxygen species (ROS) such as H2O2 are released. ROS are known to lead to oxidative stress and a variety of diseases in the human body, so developing an understanding of this uncoupling is important. It is hypothesized that a catalytic Fe–OOH intermediate is the site of bifurcation between H2O and H2O2 formation. This hydroperoxy intermediate is ubiquitous in heme-containing enzymes including cytochrome c peroxidase, heme oxygenase, and prostaglandin H synthase. In all cases, proper proton delivery is necessary for O–O bond cleavage. Mutagenesis studies of P450cam have made it clear that the role of protons and H-bonding are important in understanding selectivity, but there is debate as to how the conserved residues prevent uncoupling. Additionally, the canonical mechanism involves one proton addition to either the proximal or distal oxygen atom of the Fe–OOH intermediate to yield H2O2 or H2O, respectively, but there is no direct evidence of this stoichiometry. Our preliminary data suggests that the desired distal protonation may involve a higher dependence on protons. Therefore our goal is to study what governs the selectivity between formation of H2O or release of H2O2 from this critical Fe–OOH intermediate. Our work in this proposal seeks to understand the selectivity of H2O and H2O2 formation from heme enzymes utilizing a simple model system. Synthetic analogues provide the advantage of allowing us to systematically vary and control structural entities, enter the catalytic cycle in new places, and independently synthesize intermediates. Therefore, we propose to study the selectivity of O2 activation by Fe-porphyrin catalysts proceeding through the same Fe–OOH intermediate. First, we will explore how various reaction conditions (concentration, pKa, and structure of the acid) affect selectivity in the catalytic oxygen reduction reaction (ORR). Secondly, we will study a variety of catalysts with varied H-bonding motifs to better understand how the residues in an active site may influence selectivity. Additionally, we will explore the non-catalytic reactivity of the Fe–OOH intermediate to gain independent measures of the relative rates of H2O and H2O2 formation under varied conditions. Ultimately, our goal is to understand how the reaction conditions and H- bonding networks affect H2O versus H2O2 selectivity in a model system. This understanding will provide insight into how enzymes can control the reactivity of the critical Fe-hydroperoxy intermediate to minimize ROS formation in a variety of heme-containing active sites.