Abstract
Describing dynamics of proton transfers in proteins is challenging, but crucial for understanding processes which use them for biological functions. In cytochrome bc1, one of the key enzymes of respiration or photosynthesis, proton transfers engage in oxidation of quinol (QH2) and reduction of quinone (Q) taking place at two distinct catalytic sites. Here we evaluated by site-directed mutagenesis the contribution of Lys251/Asp252 pair (bacterial numbering) in electron transfers and associated with it proton uptake to the quinone reduction site (Qi site). We showed that the absence of protonable group at position 251 or 252 significantly changes the equilibrium levels of electronic reactions including the Qi-site mediated oxidation of heme bH, reverse reduction of heme bH by quinol and heme bH/Qi semiquinone equilibrium. This implicates the role of H-bonding network in binding of quinone/semiquinone and defining thermodynamic properties of Q/SQ/QH2 triad. The Lys251/Asp252 proton path is disabled only when both protonable groups are removed. With just one protonable residue from this pair, the entrance of protons to the catalytic site is sustained, albeit at lower rates, indicating that protons can travel through parallel routes, possibly involving water molecules. This shows that proton paths display engineering tolerance for change as long as all the elements available for functional cooperation secure efficient proton delivery to the catalytic site.
Highlights
Proton translocation across energy conserving membrane is crucial for generation of proton motive force
The quinol oxidation and quinone reduction sites can be located in two separate enzymes, or they can be assembled within one enzyme
The sequential reduction of quinone to quinol through a semiquinone intermediate (SQi) is associated with an uptake of two protons from the mitochondrial matrix or cytoplasm [9,10]
Summary
Proton translocation across energy conserving membrane is crucial for generation of proton motive force. The quinol oxidation and quinone reduction sites can be located in two separate enzymes (bacterial examples [4]), or they can be assembled within one enzyme The latter case concerns cytochrome bc, a key component of many photosynthetic and respiratory systems including mitochondrial respiration [5,6]. The sequential reduction of quinone to quinol through a semiquinone intermediate (SQi) is associated with an uptake of two protons from the mitochondrial matrix or cytoplasm [9,10]. It follows that a complete reduction of one quinone molecule at the Qi site requires oxidation of two quinol molecules at the Qo site. This secures functional connection of the two Qo and two Qi sites in the dimer
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