Finding the E-Channel Proton Loading Sites by Calculating the Ensemble of Protonation Microstates

The aerobic electron transfer chain builds a proton gradient by proton coupled electron transfer reactions through multiple proteins. Complex I is the first enzyme in this chain. On transfer of two electrons from NADH to quinone four protons are pumped from the N- (negative, higher pH) to the P- (positive, lower) side. Protons move pthrough three linear antiporter paths, with a few amino acids and waters providing the route and the E-channel, which is a complex of competing paths, with clusters of interconnected protonatable residues. Proton loading sites (PLS) are residues that transiently bind protons as they are transported from N- to P-compartments. PLS can be individual residues or extended clusters. The program MCCE uses Monte Carlos sampling to analyze the E-channel proton binding in equilibrium with individual Molecular Dynamics snapshots from trajectories of Thrmus thermuphillus Complex I in the apo, quinone or quinol bound states. At pH 7, the five E-channel subunits (Nqo4, Nqo7, Nqo8, Nqo10, and Nqo11) have accepted >25,000 protonation microstates, each with different residues protonated. The microstate explosion is tamed by analyzing interconnected clusters of residues along the proton transfer paths. A proton is bound and released to five coupled residues in a cluster on the protein N-side and to six coupled residues in the protein center. Loaded microstates bind protons to sites closer to the P-side in the forward pumping direction. MCCE microstate analysis shows the strong coupling of proton binding amongst individual residues in the two PLS clusters.