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

点击翻译按钮获取中文摘要

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 1.5Å resolution structure of this mutant, has led to the surprising discovery that the second proton channel, which was 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.