Lower Protein Stability Does Not Necessarily Increase Local Dynamics

Overall protein stability is thought to have an important impact on the millisecond time scale dynamics modulating enzyme function. In order to better understand the effects of overall stability on the substructure dynamics of mitochondrial cytochrome c, we test the effect of a destabilizing L85A mutation on the kinetics and equilibrium thermodynamics of the alkaline conformational transition. The alkaline conformational transition replaces the Met80 ligand of the heme with a lysine residue from ω-loop D, the heme crevice loop, consisting of residues 70-85. Residues 67-87 are the most conserved portion of the sequence of mitochondrial cytochrome c, suggesting that this region is of prime importance for function. Mutations to ω-loop D affect the stability of the heme crevice directly, modulating the pK_app of the alkaline transition. Two variants of yeast iso-1-cytochrome c, WT∗/L85A and WT∗/K73H/L85A, were prepared 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. The kinetics of the alkaline transition were measured by pH-jump stopped-flow methods. 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. Contrary to the expectation that dynamics around the heme crevice would be faster for the less stable WT∗/K73H/L85A variant, based on the behavior of psychrophilic versus mesophilic enzymes, 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.