As the terminal enzyme in the respiratory chain, cytochrome c oxidase (CcO) facilitates the maintaining of a transmembrane proton gradient by converting molecular oxygen to water and pumping protons across the membrane. This proton gradient is used by the enzyme ATP synthase to produce the energy storage molecule ATP. Among the most pressing open questions regarding CcO is how the redox state of its catalytic center controls proton uptake through one of its proton channels, the K-channel, and the location of proton release on the opposite side of the membrane. To elucidate these questions, an expression system was developed in Part I of this thesis enabling the background-free fluorescence labeling of Paracoccus denitrificans CcO, which is homologous to the human enzyme. Subsequent conjugation of CcO with environmentally sensitive fluorophores allowed for the investigation of nanoenvironmental parameters on localized sites on the protein surface in different enzymatic states. Steady-state and time-resolved spectroscopy techniques such as time-correlated single-photon counting (TCSPC) allowed for the detection of conformational changes, rearrangements of the local hydrogen bonding network, and changes in the hydration shell around the K-channel entrance, depending on the redox state of the enzyme. The combined results yield a model, in which the catalytic center of CcO exhibits nanoenvironmental control over the proton uptake surface above the K-channel. There, locally defined increases in conformational flexibility in water dynamics facilitate conditions putatively beneficial for the uptake of protons, which are subsequently translocated through the K-channel to the enzyme’s core. In the second part of this thesis, a novel tracking-free analysis method was developed for the determination of diffusive modes in single-molecule video microscopy. This method distinguishes itself from single-particle tracking (SPT) based techniques in its generation of step length distributions (SLDs), and thus diffusivities, from particle positions without relying on assumptions about the identity of particles as the movie progresses, are a major source of error in SPT. The herein developed method, Diffusion Analysis of Nanoscopic Ensembles (DANAE), however, generates correct SLDs even when faced with high diffusivities, high particle densities, long exposure times, as well as changes in the detectability of particles due to their photo-state, and changes imposed by ageing effects during the course of the microscopic movie, as shown through Monte Carlo Simulations (MCSs). Finally, a two-color derivation of the method (2cDANAE) is presented, which produces SLDs exclusively in case of co-diffusion between the microscopic species in each channel and can thus be used to determine the interaction of biomolecules with spectrally different fluorescence labels in multichannel single-molecule microscopy experiments.
