Exploring the mycobacterial respiratory supercomplex with Fourier-transformed electrochemistry and cryogenic electron microscopy

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Tuberculosis (TB) is a devastating disease caused by the bacteria Mycobacterium tuberculosis and is one of the top 10 causes of death worldwide. Current treatments require a cocktail of antibiotics taken over an extended period with unpleasant side-effects; drug resistance threatens to render even these treatments ineffective. New anti-TB drugs, such as bedaquiline and Q203, target enzymes in the bioenergetic system of TB. This project will focus on delivering a new complementary toolkit to deconvolute the precise chemical mechanism via which these new anti-TB drugs function in order to aid the rational design of new families of antibiotics. The student will combine Fourier-transformed electrochemical methods with structural techniques to probe the kinetic and thermodynamic control of the electron-transfer redox chemistry of the mycobacterial supercomplex. This key enzyme in the bioenergetic system of Mycobacterium tuberculosis takes electrons from menaquinol, using these to convert O2 to water and transducing the released free-energy into the proton-motive force that powers the cell. The mycobacterial supercomplex enzyme is a complex bioinorganic catalyst, it has a set of metal cofactors: heams, Cu centres, and an iron-sulphur cluster, which are used to move electrons in a controlled fashion. It is the unique structure of this enzyme that enables the design of selective drugs that will kill Mycobacterium tuberculosis while not disrupting the equivalent human enzymes. The precise chemistry of the reaction mechanism is not understood and requires in-depth mechanistic study to deconvolute the thermodynamic and kinetic control and map precisely how catalysis is inhibited by drugs such as Q203. The student will use cryo-EM to explore enzyme structure and identify drug binding sites, i.e. precisely where is the antibiotic targeting? What is the orientation of the drug molecule relative to the metal binding sites? Fourier-transformed electrochemistry will be used to study how electrons are moved through the redox-active cofactors that transmit electrons, and to unpick the inhibition mechanism, i.e. how does the drug binding shutdown catalysis? This will be the first time these complementary techniques of cryo-EM, which images biological structures frozen in the vitreous ice state, and Fourier-transformed electrochemistry, which isolates current from cofactor redox chemistry, have been combined to deconvolute antibiotic structure-function chemistry. Objectives-Using an already-prepared strain of Mycobacterium smegmatis (a harmless and fast growing model organism for TB), isolate the enzyme in a catalytically active state.-Use cryo-EM to explore different preparations of the enzyme with the in-house York Glacios electron microscope. -Apply Fourier-transformed electrochemistry to see how electrons move through the enzyme to sustain catalysis.-Combine data from the two approaches to generate quantitative models for how the enzyme functions, using tools from Marcus theory and statistical mechanics.