Abstract

Overall protein stability is thought to have an important impact on the local dynamics modulating enzyme function. In order to better understand the effects of overall stability on local dynamics in mitochondrial cytochrome c, we test the effect of a destabilizing Leu85Ala mutation on the dynamics of the tier 0 alkaline conformational transition involving conformational changes on the ms timescale. Elucidating further understanding into protein dynamics has significant relevance to health, as alternative protein conformers are associated with many human diseases. The alkaline conformational transition replaces the Met80 ligand on the heme with a lysine residue from Ω-loop D, the heme crevice loop, consisting of residues 71-85. Residues 67 to 87 are the most conserved portion of the sequence of mitochondrial cytochrome c, suggesting this region is of prime importance for function. Mutations to Ω-loop D affect the stability of the heme crevice directly, modulating the pKapp of the alkaline transition. Two variants of yeast iso-1-cytochrome c, WT∗/L85A and WT∗/K73H/L85A, were over-expressed in E. coli and purified for these studies. Guanidine-HCl unfolding monitored by circular dichroism and pH titrations at 695 nm, respectively, were used to study the thermodynamics of global and local unfolding of these variants. Dynamics of the alkaline transition were measured by pH-jump stopped-flow methods. Contrary to the expectation that dynamics around the heme crevice would be faster for the less stable WT∗/K73H/L85A variant, they were similar to those for a variant without the L85A mutation. In fact, below pH 7, the dynamics of the WT∗/K73H/L85A variant were slower. Gated electron transfer techniques using bis(2,2',2”-terpyridine)cobalt(II) as a reducing reagent were implemented to measure the heme crevice dynamics for the WT∗/K73H/L85A variant.

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