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，要么作为单个原子，要么作为簇的一部分，在细菌、植物和动物王国中扮演着各种维持生命的角色。许多酶利用金属的氧化态进行氧化还原循环。金属蛋白参与的基本生物学过程包括电子储存和传递、氧结合、储存和活化、底物运输、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的生命至关重要)、固氮酶和亚硝酸盐还原酶(鉴于它们在氮循环中的中心地位而必不可少)、氢酶(产生分子氢--未来有可能成为替代能源的有吸引力的候选者)，催化涉及受控地将电子和质子输送到底物被利用的活性部位。虽然我们对有效电子转移中涉及的因素的了解相对较好，但我们对长距离和匹配时间尺度上的质子转移的理解严重受限。在亚硝酸铜还原酶中，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置之间的受控电子转移，这必须伴随着质子的快速可用。通过对从两个不同微生物物种和大量突变体中分离的这种酶的原子分辨结构的广泛分析，我们表明电子传递受到微妙构象变化(CCET)的调节，在底物结合后，我们将其描述为活性部位周围的“传感器和信号”环。尽管我们知道质子是通过我们也确定的质子通道传递到结合在活性中心的底物上的，并且His254在其中发挥核心作用，但关于控制和调节其传递的结构因素尚不清楚。我们先前已经证明，H245F取代破坏了该通道中的水H键网络，但由于T2Cu催化位中锌的存在，无法将这与催化活性的任何影响联系起来。在过去的几周里，我们成功地将铜整合到了这个突变体中。活性测量加上该突变体的新的1.55？分辨率结构，导致了一个令人惊讶的发现：到目前为止，第二个质子通道被认为只有在高pH时才被激活，它对生理pH下的质子输送有很大贡献。对我们的NIR的0.9ä分辨率结构中氢原子位置的初步分析表明，实验上在该结构中可以看到大约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种的酶制剂，其晶体结构表明其在催化T2Cu上完全没有占据。这些酶和突变体的原子分辨结构的可用性，以及这些系统的适应性，以便通过定向突变进一步操纵，这为利用动力学、生物物理和电化学方法解决生物学中知之甚少的PCET、CCET和CGET过程的问题提供了一个理想的机会。以上概述的研究将使我们对影响生命维持过程的氧化还原酶机制的基本过程的理解发生阶段性变化。从这些研究中得出的总体原则，旨在了解电子、质子和底物传递、调节和利用的控制，也将与联合王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.