Structures of intermediates in cytochrome c oxidase

Cytochrome c oxidase (CcO) plays a central role in mitochondrial energetics. It is the final electron acceptor in the respiratory chain of mitochondria, which catalyzes the four-electron reduction of oxygen to water and harnesses the chemical energy to pump four protons across the inner mitochondrial membrane to establish an electrochemical proton gradient that is used to synthesize ATP. In humans, CcO activity is intricately regulated by a variety of cellular effectors, such as hormones, adenine nucleotides and cytochrome c. Despite extensive studies, the mechanisms by which the oxygen reduction reaction carried out by CcO is coupled to proton translocation and the activity of the enzyme is regulated by cellular effectors remain poorly understood. It creates a critical barrier hindering the development of effective therapeutic strategies against the related diseases. The two major aims of this project are to (i) determine the 3D structures of key oxygen intermediates of CcO using time-resolved serial femtosecond X-ray crystallography (SFX), thereby delineating the protein conformational changes associated with the oxygen reduction reaction occurring in the active site of the enzyme, and (ii) determine the 3D structures of CcO in complex with cellular effectors, including thyroid hormones, steroid hormones, ADP/ATP, and cytochrome c, using single-particle cryogenic-electron microscopy (cryo-EM), thereby deciphering the allosteric protein conformational changes critical for the cellular regulatory mechanisms. The structural studies will be complemented by activity assays and spectroscopic measurements to establish structure and function relationships of the enzyme. This project, built on our extensive experience in spectroscopic and structural studies of CcO, as well as our strong preliminary data in structural determination of CcO with cryo-EM, will uncover paradigms that greatly enhance our understanding of this important enzyme essential for life.