Center for Biological Electron Transfer and Catalysis (BETCy)

Description of Overall Proposal: We propose to investigate the mechanisms and structural basis governing electron bifurcation, electron-ion coupling, and catalysis in model pathways and enzymes towards developing modular biochemical conversions for the production of hydrocarbon and hydrogen biofuels. This understanding will be used to integrate new enzyme functionalities into diverse microbial physiologies as a tunable biochemical approach for controlling energy transduction, and will begin to develop experimental models for understanding the complex framework of electron distribution among diverse redox reactions. We will develop and utilize a collective knowledge base on metalloenzyme structure-function that enables re-tuning of catalytic sites, electrontransfer thermodynamics and evolution of new chemical reactions involving hydrogenases and nitrogenases. The ultimate contribution of this EFRC will be to re-design redox metalloenzymes and electron-transfer components of biochemical metabolic networks towards implementing new solutions for microbial conversion of feedstocks into biofuels. The emphasis of the EFRC will be focused on gaining a fundamental understanding of molecular mechanisms controlling bifurcation and electron flow in biological systems of energy relevance. The successful outcomes will enable the tailored re-design of the biological systems and enzymes to control matter and energy at the level of electrons and molecules in order to provide the foundations to create new energy technologies. Program thrusts will address the following biochemical molecular mechanisms in two complementary areas: Electron Transduction Networks (ETN) and Multienzyme Complexes& Enzymes (MCE): 1) Bifurcation reactions that increase the efficiency of electron transfer to end-product formation by coupling energy intensive redox reactions to allow reduced pyridine nucleotides to participate in production of a wider range of reduced energy relevant molecules. This thrust area will prove the hypothesis that electron bifurcation is a new form of energy conservation affording chemical reactions far away from equilibrium. a. Characterize representative and uncover novel bifurcating mechanisms including FeS/Flavin-containing NFN-I and NFN-II enzymes and bifurcating hydrogenases, and their coupling to any coenzyme A dependent carbon metabolic reactions b. Define the structural basis for redox potential inversion in flavoproteins-coupled reactions without requiring ATP hydrolysis c. Characterizing subunit interactions using x-ray crystallography and mass spectrometry techniques to uncover the contributions of those interaction on modulation catalytic properties and bifurcation 2) Coupling of electron transfer reactions to ATP hydrolysis in ways that increase the efficiency of the production of reduced biofuel products. a. Control of electron flux to nitrogenase and the role of Rnf and Fix electron transfer components and bifurcation mechanisms b. Role of electron flux in hydrocarbon or hydrogen in production in Mo-nitrogenase site-specific amino acid variants and alternative nitrogenases in model organisms Azotobacter vinelandii, Rhodopseudomonas palustris, and Pyrococcus furiosus c. Mechanism of ATP coupled intermolecular and intramolecular electron transfer reactions in nitrogenase directed at hydrocarbon and hydrogen production 3) Energy conservation and coupling of bifurcating and other electron transfer reactions to the formation of ion gradients across the membrane a. Characterize model mechanisms including membrane-bound H2-evolving, ionpumping ferredoxin-dependent NiFe-H2ase and homologous H2-evolving (and formate evolving) formate H2 lyase, H2ase/FDH, and carbon monoxide dehydrogenase complexes b. Investigate coupling of gradients to energy conserving reactions including ATP formation 4) Internal control of redox reaction thermodynamics through modulating redox potentials of metalloactive sites and proton donor / acceptor groups a. Define the structural determinants of metal cluster site midpoint potential as it contributes to the thermodynamics of enzymatically catalyzed redox reactions b. Define the role of proton donor/acceptor groups in proton coupled electron transfer reactions as it contributes to the thermodynamics of enzymatically catalyzed redox reactions c. Implement gene shuffling and protein design strategies for control of oxidationreduction reactions d. Refine design principles in the control of electron flow in model organisms e. Develop predictive models for directionality of enzymatically catalyzed redox reactions as a function of active site metal cluster environment 5) The role of enzyme modularity in the control of electron flow of bifurcation, energy conservation, and catalysis, and how this can be used in the design and engineering of tailored redox enzyme complexes and control of electron transfer pathways a. Modify the electron flow pathways to produce reduced products from NADH and NADPH rather than ferredoxin in model organisms b. Investigate how new carbon metabolic pathways, with and without bifurcation, alter the levels of reduced product formation