Microscopic basis for kinetic gating in cytochrome c oxidase: insights from QM/MM analysis† †Electronic supplementary information (ESI) available: Benchmark results for the DFTB3 approach for the copper site and the doubly protonated glutamate are included; validation of DFTB3/MM interaction using several relevant models is also included. Additional PMF results obtained with a different variant of DFTB are included to demonstrate that the qualitative trends are robust. Comparison of PMF and microscopic pK a calculations is included as further validation. Other materials include the isomerization of the cis/trans conformers of the Glu286 side chain, proton transfer PMF from Asp132 to the “serine zone”, and additional snapshots from various simulations. See DOI: 10.1039/c4sc01674b Click here for additional data file.

Understanding the mechanism of vectorial proton pumping in biomolecules requires establishing the microscopic basis for the regulation of both thermodynamic and kinetic features of the relevant proton transfer steps. For the proton pump cytochrome c oxidase, while the regulation of thermodynamic driving force for key proton transfers has been discussed in great detail, the microscopic basis for the control of proton transfer kinetics has been poorly understood. Here we carry out extensive QM/MM free energy simulations to probe the kinetics of relevant proton transfer steps and analyze the effects of local structure and hydration level. We show that protonation of the proton loading site (PLS, taken to be a propionate of heme a3) requires a concerted process in which a key glutamic acid (Glu286H) delivers the proton to the PLS while being reprotonated by an excess proton coming from the D-channel. The concerted nature of the mechanism is a crucial feature that enables the loading of the PLS before the cavity containing Glu286 is better hydrated to lower its pK a to experimentally measured range; the charged rather than dipolar nature of the process also ensures a tight coupling with heme a reduction, as emphasized by Siegbahn and Blomberg. In addition, we find that rotational flexibility of the PLS allows its protonation before that of the binuclear center (the site where oxygen gets reduced to water). Together with our recent study (P. Goyal, et al., Proc. Natl. Acad. Sci. USA, 110:18886-18891, 2013) that focused on the modulation of Glu286 pK a , the current work suggests a mechanism that builds in a natural sequence for the protonation of the PLS prior to that of the binuclear center. This provides microscopic support to the kinetic constraints revealed by kinetic network analysis as essential elements that ensure an efficient vectorial proton transport in cytochrome c oxidase.