Transient and Stable Macromolecular Complexes Formed by Denitrifying Enzymes

Lead Research Organisation: University of Liverpool
Department Name: Institute of Integrative Biology


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.

Technical Summary

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.

Planned Impact

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.


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Description 1. X-ray laser experiments at SACLA provide evidence of oxygen binding in Nitrite reductase

A collaboration between researchers at the University of Liverpool, UK and the RIKEN SPring-8 Centre, Japan have revealed how oxygen binds at the catalytic centre of a pivotal enzyme of the denitrification step of the nitrogen cycle where ATP formation is coupled to the reduction of nitrate rather than the reduction of oxygen. Copper nitrite reductases (CuNiR) normally reduce nitrite to nitric oxide as part of the denitrification process but some CuNiRs have oxidase activity that also allows them to reduce oxygen.

There is an extensive body of mechanistic and structural data focussed on the nitrite reductase activity of CuNiRs but reports of their interaction with oxygen have not been possible so far despite much efforts made at the best crystallographic beamlines at most intense synchrotron sources. Unfortunately, in just the same way that X-ray exposure should be limited in humans, X-ray exposure needs to be limited in the study of protein crystals. The very high intensity of synchrotron generated X-rays can result in reduction of metal centres and damage the protein during data collection, so the structure determined is not that of the resting state. Using the new type of X-ray sources, X-ray lasers that produce ultra-fast X-ray pulses of approximately 10 femtoseconds in duration diffraction data have been collected in a 'time frozen'state before any damage can occur through vibration/rotation of individual atoms in a large protein molecule. The long-standing collaboration between the Molecular Biophysics Group, University of Liverpool and the SR Life Sciences Instrumentation Unit, RIKEN SPring-8 Centre allowed for CuNiR data to be collected at SACLA. Surprisingly, Instead of the usual water molecule bound to the active copper site, seen using conventional data collection techniques, an oxygen molecule was instead observed in a unique end-on binding mode. This finding provides an insight into how CuNiRs, that two decades ago were shown to share structural similarity of their active site with superoxide dismutase can carry out the 2-electron reduction of oxygen to hydrogen peroxide with a superoxide intermediate.

The PhD student, Thomas Halsted, who performed the work and who spent two years at the RIKEN SPring-8 centre based in Dr Masaki Yamamto's group, said that he feels fortunate to have the privilege of working at the pioneering X-ray laser facility which has allowed this very elusive observation. Dr Tetusya Ishikawa, Director of Harima campus said that RIKEN-Liverpool partnership has been a very special partnership allowing exchange of scientists and PhD students. Prof Samar Hasnain who led the Liverpool team said that the dream of obtaining 'damage free' high resolution structures of redox enzyme has been realised 20 years after the first Nobel prize awarded for F1-ATPase structure using synchrotron radiation to John Walker. It is not unrealistic to expect a similar recognition for an XFEL associated science in the coming decade.

The paper "An unprecedented dioxygen species revealed by serial femtosecond rotational crystallography in copper nitrite reductase" is published in IUCrJ and can be found here (

There are only two X-ray laser facilities in operation, one at Stanford, USA called LCLS and other SACLA XFEL at the RIKEN SPring-8 Centre in Japan.
2. Identification of a tyrosine switch in copper-heme nitrite reductases We provide the first structural information of ligand bound structures of a cytochrome-fused copper nitrite reductases, several of which have been identified a decade ago. Structural characterization of the first such enzyme from Ralstonia pickettii was reported five years ago (Nature 496, 123-6 (2013)) but attempts to observe substrate/product/inhibitor bound structures have remained unsuccessful. The structure reported in 2013 by us had shown that the regular substrate access channel used by all 2-domain CuNiRs (Science 304, 867-870 (2004), PNAS 102, 12041-12046 (2005), J Mol Biol 378, 353-361 (2008), Nature 462, 117-120 (2009), IUCrJ 3, 271-281 (2016)) was closed and that a tyrosine in the active site may make the catalytic copper unavailable for binding of substrate. We show that nitric oxide activates the tyrosine in RpNiR resulting in it rotating away from the substrate binding site.

Despite the recognition that tyrosine may be redox activator in catalysis (FEBS Lett. 2012 586, 596-602 (2012)), there are still very few cases where tyrosine has been shown to be used in redox catalysis (e.g. J. Am. Chem. Soc. 136, 14039-14051 (2014) Nature 543, 131-135 (2017). We demonstrate directly through a high-resolution structure of a catalytic Cu deficient enzyme (T2D Cu enzyme) that the status of Tyr323 is not controlled by T2Cu or its redox chemistry nor through the binding of NO to heme. Our data suggest that Tyr323 switch is controlled by NO through proton abstraction from Tyr. The insight gained here for the use of tyrosine as a switch in catalysis has wider implications for catalysis in biology. The use of Tyrosine as a switch in activating the redox enzyme RpNiR has wider significance as this tyrosine is found to be totally conserved in all the known cytochrome-fused Cu-NiRs.

Our manuscript also sheds important insight into the functional significance (which has been a topic of debate) of the fused cytochrome domain in this family of denitrifying enzymes. We provide evidence that it does have two roles, protection of catalytic T2Cu by Tyr 323 that forms part of the linker between cytochrome and cupredoxin domains and secondly providing electrons to the T1Cu centre for ET gated substrate reduction.
Exploitation Route Our first publication opens up a new biological role of some of the nitrite reductases - this would lead to substantial activity in the community. The second publication under consideration demonstrates the presence of a tyrosine switch which may be commonly available in a number of related enzymes.
Sectors Agriculture, Food and Drink,Manufacturing, including Industrial Biotechology

Description Japan Partnering : Damage free structures of enzymes of denitrification pathway and their complexes using SF-ROX and SFX at SACLA XFEL
Amount £50,185 (GBP)
Funding ID BB/S020055/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 05/2019 
End 04/2023