Studies Of Electron Transfer Mechanism And Function Of Mycobacterium Smegmatis Respiratory Chain Supercomplex C?₂C?₂SOD₂

Cellular respiration is a core feature in the metabolism of many organisms.During respiration,electrons are transferred from electron donors to oxygen through an electron transport chain.The energy created allows protons to be pumped across a membrane(cellular or mitochondrial).In electron transport chains,quinones and cytochrome c are two of the electron carriers that shuttle electrons to and from large macromolecular structures that are embedded in the membrane.The components that allow respiratory chains to function in the mitochondria are well characterized,but the situation is less clear and more varied in prokaryotic systems.A soluble cytochrome c pathway for electron transfer similar to that in mitochondria is commonly found in Gram-negative bacteria.Gram-positive bacteria such as Mycobacteria are devoid of a soluble cytochrome c but instead possess cytochrome c proteins that are anchored onto the membrane or have a fused cytochrome c domain to mediate electron transfer between two of the major complexes,which are referred to as C? and C?.Structures of eukaryotic respiratory supercomplexes have been reported,but cytochrome c is not visible in any of these structures.Thus,a complete pathway for electron flow has not yet been visualized.C?-C? supercomplexes have been isolated from Mycobacterium smegmatis,Corynebacterium glutamicum,and Mycobacterium tuberculosis and shown to couple quinol oxidation to oxygen reduction without an external electron shuttle,suggesting that the flow of electrons is internalized in this type of complex.The determination of the structure of this complex reveals a path for electron transfer between the subunits of these supercomplexes.In current study,we have isolated functional supercomplex C?2C?2SOD2 from Mycobacterium smegmatis,and determined a 3.5 A three dimensional structure by single-particle electron cryo-microscopy.The structure allows the complete visualization of 20 subunits that associate to form the complex.Central to the supercomplex is a C? dimer that is flanked on either side by individual C? subunits.Fused c-type cytochrome domains bridge and mediate electron transfer from C? to C?.The respiratory supercomplex in Mycobacteria reveals cofactors positioned at distances that permit electron tunneling,enabling direct intrasupercomplex electron transfer from menaquinol to oxygen without the need for a separate cytochrome c electron shuttle.The structure also reveals three previously unidentified associated subunits that contribute to the stability of the supercomplex and the presence of superoxide dismutase(SOD),which may be responsible for the detoxification of superoxide formed by C?.The presence of a bound SOD to the respiratory supercomplex suggests a mechanism of mycobacterial resistance against exogenous and endogenous oxidative stress in macrophages and host immune responses.Oxygen consumption experiment based on protein level conducted in vitro and molecular docking experiment not only confirmed that the antituberculosis drug molecule Q203 in clinical phase ? has very similar inhibition efficiency against Mycobacterium smegmatis Mycobacterium tuberculosis,and also pointed out the Q203 binding site in the supercomplex,indicating that the current structure can be used to further optimize the Q203 and develop more effective drug molecules based on current structural foundation.