The role of human cytochrome c, HuCytc, lysine 72 in heme alkaline conformational transition has been poorly defined. It has also been thought to not act as a binding ligand as indicated by work with horse cytochrome c. Cytc has the ability to interact with cardiolipin containing vesicles. During this interaction, alternative lysine bound conformers are formed which are thought to inhibit peroxidase activity, thus making it important for understanding which residues act as alternate ligands in this transition. Here we present evidence that K72 in HuCytc may in fact act as an alternate heme ligand during the alkaline conformational transition. Using a Hu K72A mutant, we see only minor stabilization of the global structure, monitoring α-helical unfolding via circular dichroism (CD) and stepwise addition of guanidine denaturant. Shifts in stability of a global nature with a single amino acid mutation are uncommon so this is to be expected. However looking at the alkaline conformational transition with a stepwise pH titration, we see a shift in apparent pKa from 9.54±0.03 for WT to 10.00±0.13 for K72A. Our current interpretation of this shift is native state (M80 bound) stabilization caused by K72 no longer being present to displace M80. We tested this further by stopped-flow pH Jumps monitored at 406 nm for changes in heme ligation, which clearly showed a distinct change in population of alkaline conformers between WT and K72A HuCytc over a range of pH values, indicative of K72-heme ligation for WT.

Role of lysine 72 in human cytochrome c alkaline conformational transition

249th National Meeting (2015)

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