Transient and Stable Macromolecular Complexes Formed by Denitrifying Enzymes

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 - a candidate for a future alternative energy source), catalysis involves the controlled delivery of electrons and protons to the active site where substrate is utilised.

The proposal build on close collaboration between the applicants and the RIKEN group (Japan) where they collectively have made major contributions in the field of denitrification and have provided significant advances in our understanding of complex processes that are involved in biological mechanisms of metalloenzymes. Our combined approaches have allowed us to build detailed three dimensional structural movies of catalysis in the crystalline state, thereby providing detailed insight into enzyme reaction chemistry. Our recent determination of structures for the membrane proteins, nitric oxide reducatses (Tosha & Shiro, RIKEN) together with the atomic resolution structure of the tethered cytochrome-Cu1-Cu2 nitrite reductase (Antonyuk, Eady & Hasnain, UoL) puts us in a very strong position to establish an integrated structural-mechanistic biology programme. This programme is aimed at understanding complex mechanisms of redox control, regulation and communication in globally important biological systems.

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 nitric oxide. New methods and approaches developed in this programme (e.g. development of laboratory-based size-exclusion chromatography-small angle X-ray scattering with dynamic light scattering (SEC-SAXS-DLS) for studying protein complexes) will have broad relevance to our capabilities for studying protein complexes. These new capabilities and the scientific outcome will have significant impact on structural-mechanistic biology and keep the UK at the forefront of global effort in this important field.

This proposal brings together a multi-disciplinary team with a leading international partner to address a serious gap in our knowledge of some of the fundamental processes that underpin catalysis in redox enzymes. We will pursue an integrated 4 year programme drawing on the unique collaborative expertise formed by the applicants with expertise in membrane crystallography and enzymology (RIKEN) and kinetic studies, metalloenzyme crystallography and SAXS (Liverpool). We will provide important new insight into electron/proton transfer processes involved in bacterial NO respiration, both at the individual component levels catalysing its formation (NiR) and removal (NOR), and by extending our analysis towards more integrated understanding of component enzyme complexes of the NO respiratory pathway.

We have invested significantly in obtaining important supporting data for this proposal. Key is (i) our recent realisation of the cNOR and qNOR structures; (ii) discovery of an expanded family of more complex NiR proteins and atomic resolution structure of a cytochrome-Cu1-Cu2NiR tethered complex; (iii) our supporting molecular biology/biochemical studies that enables expression and mutagenesis of all of the target proteins; (iv) expression and purification of membrane qNOR proteins in active form from two organisms, one at Liverpool and one in Japan and (v) continued development of the pioneering Liverpool's SAXS facility into a SEC-SAXS-DLS (size-exclusion chromatography-small angle X-ray scattering-dynamic light scattering) facility. The team of applicants has advanced work to the point that a new phase of experimental investigation will provide transformative new insight into complex electron and proton transfer mechanisms and channelling of the cytotoxic NO radical.

Beneficiaries. The beneficiaries of the research programme are mainly academic within the international scientific community, but in the area of enabling technology we will seek to develop closer links with instrument manufacturers and facility providers to develop novel capabilities developed in our work e.g. extending Liverpool's SAXS capability into a SEC-SAXS-DLS (size-exclusion chromatography-small angle X-ray scattering-dynamic light scattering) facility that will opened to wider academic community. We already engage strongly with instrument manufacturers, assisting them in the authoring of specialised application notes and adapting instruments for new capabilities by hosting industrial colleagues in our groups to assist in technology development. We will continue to operate in this way with the explicit aim of developing wider appreciation of our novel laboratory-based combined SEC-SAXS approach under development with both academic and commercial users.

Internationalisation. The programme presents an excellent opportunity for UK scientists to develop strong programmes with a leading research centre in Japan. This will enhance University and BBSRC links with key Japanese researchers, enable exchange of research scientists (PDRAs, associated PhD students and PIs) between the UK and Japanese laboratories and ensure skills and technology dissemination. Extended visits by the PDRA to the Japanese group will provide valuable training in specialised aspects of membrane-protein crystallography and facilitate import of the knowledge back to the UK. We will establish a series of workshops/symposia that will enable us to develop further links with Japanese groups in the area of structural biology, biophysics and mechanism thereby facilitating better integration of their expertise in the UK research programmes. We will integrate these symposia into the University of Liverpool-RIKEN partnering scheme to provide new opportunities for UK scientists to work in the Japan (e.g. PhD student exchange scheme) and vice versa. Such an agreement is in place, but we will seek to expand this programme using the networking and workshops developed in this grant award.

Outreach. We will take advantage of the framework of a green agenda and food sustainability, as part of the University of Liverpool's Food Security Network. This will be in addition to our planned lectures at regional schools and public lecture events.

Communication. We will communicate and develop our infrastructure and approaches through frequent networking events with external stakeholders through structured workshops, showcase events and industry within the University of Liverpool. In particular, we are developing new biophysical capabilities especially in the SEC-SAXS. We will hold a specific 'Integrated biophysical approaches' workshop, as part of the International year of Crystallography (2014) activity, to which we will invite academic groups, instrument manufacturers, industry groups and non-academic scientists to communicate the power of integration of crystallography with additional biophysical methods. An important aspect here will be training and scientific development of younger workers. Our PDRAs will engage in science exhibitions and public lectures such as Science and Society Lectures at Liverpool.