Infrared detection of functional waters and protonatable amino acids; solving the proton-electron coupling mechanism of cytochrome oxidase

细胞色素c氧化酶是人体供能呼吸链中的一个组成部分，负责消耗我们呼吸的大部分氧气。它利用食物的新陈代谢产物将氧气还原为水。反应释放的能量被用来驱动质子穿过它所在的膜。这种质子梯度被用来驱动ATP的形成，ATP是我们细胞内无数反应所需的能量的主要供应者。我们已经知道了很多关于酶的原子结构和催化耗氧化学的辅助因素。更难确定的是，这些反应如何导致质子运动，从而保存释放的能量。这是因为在对晶体进行X射线研究后得出的原子模型中不能看到质子，而且质子的运动路径很可能是在催化循环中发生变化的动态结构。中红外振动光谱(IR或‘FTIR’光谱)提供了一种通过测量蛋白质吸收红外光的方式的微小变化来测量蛋白质在不同状态之间变化时的变化的方法。这些红外变化被很好地分辨，以至于我们可以根据原子团的特定原子运动，或者蛋白质中特定氨基酸或辅因子的化学变化来解释它们。通过使蛋白质在对其功能重要的两种状态之间变化，我们可以通过红外光谱来‘看到’正在发生的原子变化。当晶体结构已经产生静态原子结构时，这项技术是最强大和有用的，就像细胞色素氧化酶的情况；在这种情况下，我们可以结合静态X射线结构和红外光谱的信息来描述催化的动态机制。越来越清楚的是，埋藏在蛋白质中的水分子通常在催化中发挥着与氨基酸和辅因子一样重要的作用。水在质子转移途径中的作用尤为重要。我们对细胞色素氧化酶中质子转移的耦合在原理上是如何工作的有很好的了解，但可能是缺乏监测功能水参与的能力，这阻碍了对原子机制的详细了解。近年来，用红外光谱直接测定水分子已成为可能。我们已经对细胞色素氧化酶进行了初步研究，可以清楚地看到这种结构性水分子的变化方式，这表明它在质子转移催化中发挥了作用。在这个项目中，我们的目标是监测这些水分子如何随着特定氨基酸和辅因子的变化而变化。对于许多酶来说，典型催化反应的重要步骤发生在微秒-毫秒范围内，就像细胞色素氧化酶的情况一样，我们将测量的正是这一范围的时间尺度。这项工作的总体结果将描述细胞色素氧化酶能量守恒的基本机制，这将有助于我们了解该酶如何在健康和疾病中发挥作用和故障。它还将提供对水在酶催化中的重要性的更广泛的见解。最后，这项技术本身在英国是独一无二的--它将为研究其他酶系统中的水提供基本资源，并正在开发用于医疗诊断的实际应用。

Cytochrome c oxidase is a component of the human energy-providing respiratory chain that is responsible for consumption most of the oxygen that we breathe. It uses products of metabolism of food to reduce the oxygen to water. Energy that is released by the reaction is used to drive protons across the membrane in which it resides. This proton gradient is used to drive formation of ATP, the major supplier of energy needed by the myriad of reactions within our cells. We already know a great deal about the atomic structure of the enzyme and the cofactors that catalyse the oxygen-consuming chemistry. What has been much more difficult to determine is how these reactions cause the proton movements that conserve the released energy. This arises because protons cannot be 'seen' in atomic models derived from X-ray studies of crystals and because the paths on which they travel are likely to be dynamic structures that change through the catalytic cycle. Mid-infrared vibrational spectroscopy (IR or 'FTIR' spectroscopy) provides the means measure changes in proteins as they change between different states by measuring tiny changes in the way that the proteins absorb infrared light. These infrared changes are sufficiently well resolved that we can interpret them in terms of specific atomic movements of groups of atoms, or of chemical changes of specific amino acids or cofactors within the protein. By causing the protein to change between two states that are important for its function, we can IR spectroscopy to 'see' atomic changes that are occurring. The technique is most powerful and useful when crystal structures have already produced a static atomic structure, as is the case for cytochrome oxidase; in such cases we can combine information from static X-ray structures with that from IR spectroscopy to provide a description of the dynamic mechanism of catalysis. It is becoming increasingly clear that water molecules buried within proteins often have roles in catalysis that are as central as those of amino acids and cofactors. Roles for water in proton transfer pathways are particularly important. We have a good idea of how the coupling of proton transfer in cytochrome oxidase works in principle, but it is probably the lack of ability to monitor functional water involvement that has precluded detailed understanding of the atomic mechanism. In recent years, it has become feasible with IR spectroscopy to directly measure such water molecules. We have carried out initial studies with cytochrome oxidase and can clearly see such structural waters molecules that change in a manner that suggests a role in proton transfer catalysis. In this project, we aim to monitor how these water molecules change in concert with specific amino acid and cofactor changes. For many enzymes, the important steps of a typical catalytic reaction occur on the microseconds-milliseconds range, as is the case for cytochrome oxidase, and it is this range of timescales that we will be measuring. The overall outcome of the work will provide a description the basic mechanism of energy conservation in cytochrome oxidase and this will help us understand how this enzyme functions and malfunctions in health and disease. It will also provide insights into the importance of water in enzyme catalysis more generally. Finally, the technology itself is unique in the UK -- it will provide a basic resource to study water in other enzyme systems and is also being developed for practical applied applications in medical diagnostics.