Date of Degree


Document Type


Degree Name





Marilyn Gunner

Committee Members

Nicolas Biais

Qiang Cui

Ronald Koder

Hernan Makse

Subject Categories

Biological and Chemical Physics | Biophysics


proton transfer; Grotthuss shuttling; proton loading site;proton affinity


Cytochrome c Oxidase (CcO), is the terminal electron acceptor in the membrane bound aerobic respiratory chain. It reduces O2 to water. The energy released by this reaction is stored by pumping protons from the high pH, N-side of the membrane to the low pH, P-side. The generated proton gradient provides the motive force for synthesis of ATP by the ATP synthase.

Building a proton gradient across the membrane requires that proton transport must occur along controllable proton pathways to prevent proton leakage to the N-side. It has been suggested that CcO function requires proton transfer channels in both the N- and P-sides of CcO, a connection between them that can be open and closed, and a proton loading sites (PLS) on the P-side to hold protons transiently. In this way, a proton is transferred into the protein through N-side proton channels and is held in the PLS for enough time to allow the P-side proton pathways to open and release the protons to P-side of the membrane. The A-type CcO has well established D- and K-channels to transport protons to the Binuclear Center (BNC), where the Oreduction happens, and to the PLS. The PLS in A-type CcO has been suggested to be a cluster near the propionic acids of heme a3. However, because of the complexity of the buried hydrogen bond network on the P-side, the identity of the groups in the proton exit pathway remained a mystery.

In this thesis, Multi-Conformation Continuum Electrostatics (MCCE), combined with Molecular Dynamics (MD), is applied on CcO to identify the proton exit pathway on the P-side in A-type CcO and to identify the PLS in B-type CcO and characterize proton loading/unloading mechanism. MCCE samples the water locations, polar proton positions and residue protonation states using MD snapshots and crystal structures as input. It identified a water-mediated hydrogen bond network in the A-type CcO. The inter-connections between the clusters was found to change with the redox state of the input structure revealing a complete, controllable proton transport pathway in A type CcO. A water filled cavity near Glu286 at the end of the D-channel changes hydration and thus the connection between N- and P-side channels in structures prepared in different redox states. The proton exit pathway, located on the P-side beyond the PLS, has key residues including His93, Thr100 and Asn96. A water wire in a cavity centered near Thr100, can be interrupted by a hydrophobic pair Leu225B and Ile99, which may open and close the proton exit pathway of A-type CcO.

MCCE simulation on MD trajectories and crystal structures of B-type CcO, monitored the protonation change in different imposed redox states. The PLS is identified by monitoring residues that change protonation during this redox cycle. Six residues are identified as the PLS: the heme a3 propionic acids, Asp287, Asp372, His376 and Glu126B. The analysis of the ionization state change in 136 crystal structures and trajectory snapshots suggests that there are four states for the PLS in B-type CcO: active loaded and unloaded states, in which the PLS can release and bind protons through the reaction cycle; and the locked, loaded and unloaded states, which are trapped in one protonation state. The active PLS has one proton on Asp372 and one proton that can be bound to and released from PRAa3, the propionic acid of heme a3. The locked loaded state of PLS instead has one proton on Asp372 and one proton on His376. The transition of the PLS states correlates with the changes in the hydrogen bonds amongst Asp372, His376 and PRAa3.