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，构成了蛋白质王国的很大一部分。金属蛋白本身约占基因组的30%。它们含有金属离子，或作为单个原子，或作为簇的一部分，在细菌、植物和动物王国中起着各种维持生命的作用。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过程包括电子储存和转移、二氧结合、储存和激活、底物运输、催化和激活。在许多金属酶中，如细胞色素c氧化酶（哺乳动物通过呼吸需要生命所必需的），氮酶和亚硝酸盐还原酶（鉴于它们在氮循环中的中心位置而必不可少），氢化酶（分子氢的生产者-未来替代能源的一个有吸引力的候选者），催化包括电子和质子的受控传递到利用底物的活性位点。虽然我们对有效电子转移所涉及的因素的理解相对较先进，但我们对远距离和匹配时间尺度上的质子转移的理解严重有限。在亚硝酸盐铜还原酶中，我们已经证明底物的利用伴随着电子传递和底物结合金属位点之间的受控电子转移，这必须伴随着质子的快速可用性。通过对从两种不同的微生物物种和大量突变体中分离出的这种酶的原子分辨率结构的广泛分析，我们已经表明，电子传递是由微妙的构象变化（CCET）调节的，我们将其描述为底物结合后活性位点周围的“传感器和信号”环。虽然我们知道质子通过质子通道被传递到活性位点结合的底物上，并且我们也确定了His254在其中起核心作用，但没有关于控制和介导其传递的结构因素的信息。我们之前已经证明，H245F取代破坏了该通道中的水h键网络，但由于在T2Cu催化位点存在Zn，因此无法将其与对催化活性的任何影响联系起来。在过去的几周里，我们已经成功地将铜整合到这个突变体中。活性测量与该突变体的新的1.55Å分辨率结构一起，导致了令人惊讶的发现，第二质子通道，迄今为止被认为仅在高pH下被激活，对生理pH下的质子传递有重要贡献。在我们的0.9Å近红外分辨率结构中氢原子位置的初步分析显示，在实验结构中可以看到约30%的预期氢原子。最近，我们还成功地从细胞提取物中分离出具有稳定亚硝基物质的酶制剂，其晶体结构显示在催化的T2Cu上有完全的NO占用。这些酶和突变体的原子分辨率结构的可用性，以及这些系统对定向诱变的进一步操纵的适应性，提供了一个理想的机会，可以应用广泛的程序，利用动力学，生物物理和电化学方法来解决生物学中知之甚少的PCET， CCET和CGET过程的问题。上述研究将为我们对氧化还原酶机制的基本过程的理解提供一个台阶，氧化还原酶对生命维持过程的影响。从这些研究中得出的总体原则，旨在理解电子、质子和底物传递、调节和利用的控制，也将与英国通过综合生物学方法理解生物过程的努力具有更广泛的相关性。

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.