Towards a paradigm shift in understanding of membrane-bound Nitric Oxide reductase and its complexes with the electron donor and NO-producing enzyme

About one third to one half of all proteins are oxidation/reduction enzymes or metalloproteins. It is estimated that more than one third of all proteins in nature require metals to perform their biological roles and nearly half of all enzymes must associate with a particular metal to function. These metal ions can be either a single atom or form part of a cluster, playing 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 during catalysis. 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, hydrogenases, nitrogenases and nitrite reductases, catalysis involves the controlled delivery of electrons and protons to the active site where chemical substrates are utilised. These events are often coordinated, coupled and orchestrated by structural signals that remain poorly understood in many cases due to the experimental limitations, particularly membrane proteins, that require solubilization and can be difficult to crystallize. Consequently, although the number of unique structures for membrane proteins has steadily increased since the first structure of a membrane protein in 1985, which brought the Nobel prize in 1988 to Deissenhoffer, Huber and Michel, progress has been slower than predicted. However, recent advances in cryoEM has provided a major boost to structure determination of membrane proteins, catching up the target set in 1990.

This project is built on an excellent track record of collaboration and significant underpinning data including the highest resolution structure of any NOR to provide a step change in our understanding of this important membrane metalloenzyme and its complex with NO producing enzyme (AxNiR) that has been studied in our laboratory for several years. The project would provide the first example of protein-protein complexes in catalytic turnover for NOR.

This is a challenging project but is highly achievable given our experience and our underpinning data as well as availability of high-quality proteins and several mutants. Our aim is to provide answers to many of the generic questions which are fundamental for (a) protein-protein and protein-ligand interactions, (b) substrate guidance and binding, (c) substrate utilisation with coordinated delivery of electron and proton and (d) product formation and its release. The ability of cryoEM to provide high resolution structure of a frozen solution sample of proteins will enable many of these questions to be addressed, as has been demonstrated very recently for two-component nitrogenases enzyme under turnover conditions (Science 377, 865-869 (2022)).

The applicants have an excellent track record of collaboration using cryoEM that has led to several key publications during the last 4 years. Exciting developments arising from our structural and mechanistic work on enzymes catalysing the formation of nitrous oxide by membrane bound quinol-dependent NORs now underpin this timely well-integrated programme where our complementary expertise is harnessed to maintain a world-leading position. General principles emerging from these studies will underpin our understanding of the control of redox processes in biology and protection against toxic chemical intermediates like NO. New methods and approaches that will be developed in this programme will have broad relevance to structural enzymology and keep the UK at the forefront of the global effort. The project would provide a high level of training in membrane structural biology, frontier cryoEM methodology, technology, data processing and structural refinement at high resolution.

This collaborative proposal from two leading teams who have access to latest cryoEM facilities (both at Leeds and eBIC) aims to address a serious gap in our knowledge of some of the fundamental processes that underpin catalysis in the respiratory heme-copper oxidase (HCO) superfamily. The proposal will utilise cutting edge membrane-protein cryoEM techniques in combination with well-designed mutants based on the recently obtained structures of the active dimeric form of the two qNORs, NmqNOR and AxqNOR and the highest resolution cryoEM structure of AxqNOR that we have recently obtained. We would combine these with functional studies based on NO consumption as well as N2O generation. We can start to address some of the key questions on (i) how and where qNORs bind electron donor, (ii) how is NO is converted to N2O utilising a heme-Fe catalytic unit, (iii) how electron is relayed from heme b to heme b3-Fe core, iv) where does the NO producing CuNiR bind and (v) where does NO enter and how it is delivered to the catalytic core. This aspect of NOR function would be investigated via the complex formation with the NO producing copper nitrite reductase (CuNiR) from the same organism and AxqNOR. Key to the success of this ambitious proposal is the strength of the current pilot data, e.g. (i) we have obtained highly active qNOR preparations for both the wild type and several mutants, (ii) obtained the highest resolution structure of any NOR, (iii) have established procedures for producing AxNiR and several of its mutants. Extensive mutational, functional and structural studies would be undertaken to provide important new insight for processes involved in bacterial NO respiration and catalysis.