Date of Degree


Document Type


Degree Name





Marilyn R. Gunner

Committee Members

Ronald Koder

Ranajeet Ghose

Nicolas Biais

Abhishek Singharoy

Subject Categories

Biological and Chemical Physics | Physics


Complex I, Protonation microstates, Proton transfer pathways, hydrogen bond networks


Complex I, NADH-ubiquinone oxidoreductase, is the first enzyme in the mitochondrial and bacterial aerobic respiratory chain. It pumps four protons through four transiently open pathways from the high pH, negative, N- side of the membrane to the positive, P-side driven by the exergonic transfer of electrons from NADH to a quinone. Three protons transfer through subunits descended from Mrp antiporters, while the fourth, E-channel is unique. Because of the complex possible paths thorough the many buried polar residues and lack of high-resolution crystal structure, the path for protons through the E-channel is elusive.

In this dissertation, the E-channel proton pumping pathway of Complex I is investigated by using network analysis and protonation microstates approaches. First, the path through the E-channel is determined by network analysis of hydrogen bonded pathways obtained by Monte Carlo sampling of protonation states, polar hydrogen orientation, and water occupancy. Input coordinates are derived from molecular dynamics trajectories comparing substrate oxidized, reduced (dihydro) product and no menaquinone-8 (MQ) bound. A complex proton transfer path from the N- to the P-side is found consisting of six clusters of highly inter-connected hydrogen-bonded residues. The network connectivity depends on the presence of quinone and its redox state, supporting a role for this cofactor in coupling electron and proton transfers. The N-side is more organized with MQ-bound complex I facilitating proton entry, while the P-side is more connected in the apo-protein, facilitating proton exit. Subunit Nqo8 forms the core of the E channel; Nqo4 provides the N-side entry, Nqo7 and then Nqo10 join the pathway in the middle, while Nqo11 contributes to the P-side exit.

In the second part of the thesis, a novel tool to analyze the microstates found in the ensemble of accepted states in Monte Carlo sampling is described. The distribution of protonation microstates throughout the protein is needed to develop a global pumping mechanism for proton transfer. The protonation state of residues, cofactors, and ligands defines a “protonation microstate”. Here, the protonation microstates generated in Monte Carlo sampling in MCCE are characterized in HEW lysozyme as a function of pH, bacterial photosynthetic reaction centers (RCs) in different reaction intermediates and E-channel of Complex I with oxidized, and no menaquinone-8 (MQ). The lowest energy and highest probability microstates are compared. The ΔG°, ΔH°, and ΔS° between the four protonation states of Glu35 and Asp52 in lysozyme are shown to be calculated with reasonable precision. A weighted Pearson correlation analysis shows coupling between residue protonation states in RCs and the E-channel of Complex I. The inter-cluster correlation analysis in the E-channel shows there is not much correlation between the clusters. Protonation microstates can be used to define input MD parameters and provide insight into the motion of protons coupled to reactions.