Coupled electron and proton transfer (CEPT) events are fundamental for many bioenergetic conversions that involve redox reactions. Understanding the details underlying CEPT processes will advance our knowledge of (1) how nature regulates energy conversion; (2) our strategies for achieving renewable energy sources; (3) how to cope with CEPT dysfunction diseases. Studies of the detailed mechanism(s) of CEPT in biological systems is challenging due to their complex nature. Consequently, controversies between the concerted and sequential mechanism of CEPT for many systems remain. This dissertation focuses on the bovine mitochondrial cytochrome bc1 complex. CEPT in the bc1 complex operates by a modified "Q-cycle"(1) and catalyzes electron transfer from ubiquinol (QH2), to cyt c via an iron sulfur cluster (ISC) and to the low potential hemes of cyt b, where it reduces ubiquinone (UQ). The electron transfer is coupled to the translocation of protons across the mitochondrial inner membrane, generating a proton gradient that drives ATP synthesis. Although the Q-cycle is widely accepted as the model that best describes how electrons and protons flow in bc1, detailed binding geometries at the Qo site (QH2 oxidation site) and Qi site (UQ reduction site) remain controversial. The binding geometries play critical roles in the thermodynamic and/or kinetic control of the reaction and protonatable amino acid side chains can participate in the proton transfer. The central focuses of this dissertation are molecular dynamics simulations of cofactor binding geometries near the Qo and Qi sites, calculations of the pKa values of ionizable amino acid side chains implicated in cofactor binding, especially the ISC-coordinated histidines, and implications for the proposed mechanism(s) of CEPT. For the first time, pKa values of the ISC-coordinated histidines are differentiated. The computed pKa values of 7.8±0.5 for His141 and 9.1±0.6 for His161 agree well with experiment (7.63±0.15 and 9.16±0.28). Thus, His161 should be protonated at physiological pH and cannot be the first proton acceptor in the QH2 oxidation. Water mediated hydrogen bonds between substrate models and the protein and water accessibility to the Qo and Qi sites were maintained in simulations, implying that water molecules are likely the proton donors and acceptors. / Bayer School of Natural and Environmental Sciences; / Chemistry and Biochemistry / PhD; / Dissertation;

The cytochrome bc1 complex of the mitochondrial electron transport chain accomplishes the enzymatic reaction known as the modified Q-cycle. In the Q-cycle the bc1 complex transports protons from the matrix to the intermembrane space of the mitochondria, creating the proton gradient used to make ATP. The energy to move these protons is obtained by shuttling electrons from the coenzyme ubiquinol (QH2) to coenzyme ubiquinone (Q) and the mobile cytochrome c. This well studied complex is ideal for examining molecular adaptation because it consists of ten different subunits, it functions as a dimer, and it includes at least five different active sites. The program TreeSAAP was used to characterize molecular adaptation in the bc1 complex and identify specific amino acid sites that experienced positive destabilizing (radical) selection. Using this information and three-dimensional structures of the protein complex, selection was characterized in terms of coevolution and coadaptation. Coevolution is described as reciprocal local biochemical shifts based on phylogenetic location and results in overall maintenance. Coadaptation, on the other hand, is more dynamic and is described as coordinated local biochemical shifts based on phylogenetic location which results in overall adaptation. In this study both coevolution and coadaptation were identified in various locations on the protein complex near the active sites. Sites in the pore region of cyt c1 were shown to exhibit coevolution, in other words maintenance, of many biochemical properties, whereas sites on helix H of cyt b, which flanks the active sites Qo and Qi, were shown to exhibit coadaptation, in other words coordinated shifts in the specific properties equilibrium constant and solvent accessible reduction ratio. Also, different domains of the protein exhibited significant shifts in drastically different amino acid properties: the protein imbedded in the membrane demonstrated shifts in mainly functional properties, while the part of the complex in the intermembrane space demonstrated shifts in conformational, structural, and energetic properties.