Elucidating mechanisms of proton coupled and conformationally coupled electron transfer in redox enzymes catalysis

氧化还原蛋白，包括金属蛋白，形成蛋白质王国的很大一部分。金属蛋白本身占基因组的30%。这些包含金属离子作为单个原子或作为簇的一部分，并在细菌，植物和动物王国中发挥各种生命维持作用。许多酶利用金属的氧化态来进行氧化还原循环。金属蛋白参与的基本生物学过程包括电子储存和传递、分子氧结合、储存和活化以及底物转运、催化和活化。在许多金属酶中，如细胞色素c氧化酶（通过呼吸需求对哺乳动物生命至关重要）、固氮酶和亚硝酸盐还原酶（鉴于其在氮循环中的中心位置而至关重要）、氢化酶（分子氢的生产者-未来替代能源的有吸引力的候选者），催化涉及电子和质子到利用底物的活性位点的受控递送。虽然我们对有效电子转移中所涉及的因素的理解相对较先进，但我们对长范围内和匹配时间尺度上的质子转移的理解是非常有限的。在亚硝酸铜还原酶，我们已经表明，利用基板是伴随着一个受控的电子传递和基板之间的结合金属网站，必须伴随着一个质子的快速可用性的电子转移。通过广泛的原子分辨率结构的分析，这种酶从两种不同的微生物物种和大量的突变体分离，我们已经表明，电子传递的调节微妙的构象变化（CET）在我们所描述的“传感器和信号”环周围的活性位点结合底物。虽然我们知道质子通过我们已经确定的质子通道传递到活性位点结合的底物，并且His 254在其中起着核心作用，但没有关于控制和介导其传递的结构因子的信息。我们以前已经表明，H245 F取代破坏了水的H-键合网络在这个通道中，但无法关联这与任何影响的催化活性，由于锌的存在下，在T2 Cu催化位点。在过去的几周里，我们已经成功地将Cu整合到这个突变体中。活性测量以及该突变体的新的1.55 μ m分辨率结构导致了令人惊讶的发现，即迄今为止被认为仅在高pH下激活的第二质子通道，在生理pH值下对质子传递有显著贡献。对NiR的0.9 μ m分辨率结构中氢原子位置的初步分析表明，约30%的NiR结构中氢原子的位置与质子的传递有关。的预期氢原子是可见的结构实验。最近，我们还成功地分离制备酶与稳定的亚硝酰基物种从细胞提取物，其晶体结构已揭示完全NO占用在催化T2 Cu。这些酶和突变体的原子分辨率结构的可用性，以及这些系统的定向诱变进一步操作的顺从性，提供了一个理想的机会，应用广泛的程序，利用动力学，生物物理和电化学方法的问题知之甚少PCET，CCET和CGET过程中的生物学。上面概述的研究将为我们理解氧化还原酶机制的基本过程提供一个步骤，氧化还原酶影响生命维持过程。从这些研究中得出的总体原则，旨在了解电子，质子和底物的传递，调节和利用的控制，也将是更广泛的相关性，英国的努力，通过综合生物学方法理解生物过程。

Redox proteins, including metalloproteins, form a large portion of the protein kingdom. Metalloproteins themselves form ~ 30% of a genome. These contain metal ions either as a single atom or as part of a cluster and play a variety of life sustaining roles in the bacterial, plant and animal kingdoms. Many enzymes exploit the oxidation states of metals to perform redox cycling. Fundamental biological processes in which metalloproteins participate include electron storage and transfer, dioxygen binding, storage and activation, and substrate transport, catalysis and activation. In many metalloenzymes such as cytochrome c oxidase (essential for mammalian life through respiratory requirements), nitrogenases and nitrite reductases (essential in view of their central position in the nitrogen cycle), hydrogenases (producers of molecular hydrogen - an attractive candidate for a future alternative energy source), catalysis involves the controlled delivery of electrons and protons to the active site where substrate is utilised. While our understanding of factors involved in effective electron transfer is relatively well advanced, our understanding of proton transfer over a long range and on a matching time scale is severely limited. In copper nitrite reductases, we have shown that utilisation of substrate is accompanied by a controlled electron transfer between the electron delivery and substrate binding metal sites which must accompany a rapid availability of a proton. Through extensive analysis of atomic resolution structures of this enzyme isolated from two different microbial species and a large number of mutants, we have shown that electron delivery is regulated by subtle conformational changes (CCET) in what we have described as the 'sensor and signaling' loops around the active site following the binding of substrate. Although we know that the proton is delivered to the substrate bound at the active site via a proton channel that we have also identified, and where His254 plays a central role, no information is available on the structural factors that control and mediate its delivery. We have previously shown that the H245F substitution disrupts the water H-bonding network in this channel but were unable to correlate this with any effect on catalytic activity due to the presence of Zn in the T2Cu catalytic site. During the last few weeks, we have been successful in incorporating Cu into this mutant. Activity measurements together with a new 1.55Å resolution structure of this mutant, has led to the surprising discovery that the second proton channel, which so far has been presumed to be activated only at high pH, contributes significantly to proton delivery at physiological pH. Preliminary analysis of the location of hydrogen atoms in our 0.9Å resolution structure of NiR has revealed that some 30% of the expected hydrogen atoms are visible in the structure experimentally. Recently, we have also succeeded in isolating preparations of enzyme with a stable nitrosyl species from cell extracts, the crystal structure of which has revealed full NO occupancy at the catalytic T2Cu. The availability of atomic resolution structures for these enzymes and mutants, and amenability of these systems for further manipulation by directed mutagenesis, presents an ideal opportunity to apply a wide-ranging programme utilising kinetic, biophysical and electrochemical approaches to the problem of poorly understood PCET, CCET and CGET processes in biology. The studies outlined above will provide a step-change in our understanding of the fundamental processes that underlie the mechanisms of redox enzymes, which impact on life-sustaining processes. The overall principles derived from these studies, aimed towards an understanding of the control of electron, proton and substrate delivery, regulation and utilization will also be of broader relevance to UK's effort in understanding biological processes through an integrated biology approach.