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 a series of proteins. Complex I is the first enzyme in the sequence. Here transfer of two electrons from NADH to quinone yields four protons pumped from the membrane N- (negative, higher pH) side to the P- (positive, lower pH) side. Protons move through three linear antiporter paths, with a few amino acids and waters providing the route; and through the E-channel, a complex of competing paths, with clusters of interconnected protonatable residues. Proton loading sites (PLS) transiently bind protons as they are transported from N- to P-compartments. PLS can be individual residues or extended clusters of residues. The program MCCE uses Monte Carlos sampling to analyze the E-channel proton binding in equilibrium with individual Molecular Dynamics snapshots from trajectories of Thermus thermuphillus Complex I in the apo, quinone and quinol bound states. At pH 7, the five E-channel subunits (Nqo4, Nqo7, Nqo8, Nqo10, and Nqo11) take >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 from a cluster of five coupled residues 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 identifies strongly coupled proton binding amongst individual residues in the two PLS clusters. •MCCE calculates protonation states in Complex I using MD trajectory snapshots.•Two proton loading sites (PLS) were identified in Complex I E-channel.•A proton binds to five residues N-side and six in center, forming two PLS.•MCCE microstates show proton binding in PLS clusters with strong residue coupling.•Center PLS loads protons with quinone; N-side loads more in apo or MQH2.