Exploration of the cytochrome c oxidase pathway puzzle and examination of the origin of elusive mutational effects.

Gaining detailed understanding of the energetics of the proton-pumping process in cytochrome c oxidase (CcO) is a problem of great current interest. Despite promising mechanistic proposals, so far, a physically consistent model that would reproduce all the relevant barriers needed to create a working pump has not been presented. In addition, there are major problems in elucidating the origin of key mutational effects and in understanding the nature of the apparent pK(a) values associated with the pH dependencies of specific proton transfer (PT) reactions in CcO. This work takes a key step in resolving the above problems, by considering mutations, such as the Asn139Asp replacement, that blocks proton pumping without affecting PT to the catalytic site. We first introduce a formulation that makes it possible to relate the apparent pK(a) of Glu286 to different conformational states of this residue. We then use the new formulation along with the calculated pK(a) values of Glu286 at these different conformations to reproduce the experimentally observed apparent pK(a) of the residue. Next, we take the X-ray structures of the native and Asn139Asp mutant of the Paracoccus denitrificans CcO (N131D in this system) and reproduce for the first time the change in the primary PT pathways (and other key features) based on simulations that start with the observed structural changes. We also consider the competition between proton transport to the catalytic site and the pump site, as a function of the bulk pH, as well as the H/D isotope effect, and use this information to explore the relative height of the two barriers. The paper emphasizes the crucial role of energy-based considerations that include the PT process, and the delicate control of PT in CcO.