Spectroscopic investigations of mycobacterial cytochromes

The study of bioenergetics has been built upon measurements from ex-vivo techniques. These techniques are powerful but necessarily divorced from the cellular reality in which bioenergetic systems operate. This thesis presents a non-invasive spectroscopic technique allowing in situ measurement of the electron transport chain (ETC), an essential part of the
bioenergetic system.

The approach developed here, remission spectroscopy, measures back-scattered visible-
wavelength light from the culture. Cytochromes absorb strongly in the visible region, and the absorbance depends on their redox state. They are mechanistically essential to the function of the ETC and are excellent spectroscopic handles for noninvasively measuring
bioenergetics in vivo. The system is used to explore Mycobacterium smegmatis, a safe model mycobacterium for M. tuberculosis, which is currently the worst global infectious killer.

Recently, the approval of the ATP-synthase inhibitor bedaquiline (BDQ) has focused attention on mycobacterial bioenergetics as a target space. However, the study of mycobacterial bioenergetics has been hampered by a lack of methods. Methods for making spectroscopic measurements on mycobacteria in defined growth conditions are developed, as are effectors to perturb the bioenergetic system. Preliminary experiments with in vivo spectra demonstrated that data from homologous enzymes is incompatible with mycobacterial ones. The spectral characterisation of the mycobacterial cyt bcc:aa3 supercomplex using spectroelectrochemistry addresses this. Complete analysis of this data required an integrated model of spectra and statistical thermodynamics, using computational and mathematical tools. Using the developed model provides an effective tool for measuring ΔΨ in vivo, using endogenous and direct signals in a manner that allows the measurement before and after perturbations. The foundations for future biochemistry on other cytochrome signals are established. These could be used similarly characterised the other mycobacterial cytochromes leading to a spectral-thermodynamic model of the whole mycobacterial ETC. This would be an unprecedented window into bacterial bioenergetics.