Effects of Heme Pocket Structure and Mobility on Cytochrome c Stability^†

Unfolding thermodynamics of a thermophilic cytochrome c₅₅₂ from Hydrogenobacter thermophilus (Ht cyt c₅₅₂) and its mesophilic homologue from Pseudomonas aeruginosa (Pa cyt c₅₅₁) as well as two heme pocket point mutants (Ht-Q64N and Pa-N64Q) are characterized by determination of protein stability curves (plots of unfolding free energy, ΔG, vs T). These proteins show revealing differences in heme pocket hydrogen bonding and mobility. It previously has been shown that Asn64 in Pa cyt c₅₅₁ and in Ht-Q64N interacts with the heme axial Met to fix it in a single conformation [Wen, X., and Bren, K. L. (2005) Biochemistry 44, 5225−5233]. In Ht cyt c₅₅₂ and Pa-N64Q, Gln64 does not interact with the axial Met; in these variants the axial Met samples more than one conformation [Wen, X., and Bren, K. L. (2005) Inorg. Chem. 44, 8587−8593]. Here it is demonstrated that, relative to wild type, Pa-N64Q displays enhanced stability with an increase in unfolding free energy (ΔΔG) of 7.1 kJ/mol and an increase in denaturation temperature (ΔT_m) of 8 °C. Correspondingly, Ht-Q64N is less stable than Ht cyt c₅₅₂, with a ΔΔG of −10 kJ/mol and a ΔT_m of −10 °C. Analysis of unfolding thermodynamics indicates that the net changes in stability resulting from the position 64 mutations are primarily attributable to entropic factors. For Pa-N64Q (Ht-Q64N) it is proposed that the favorable release (unfavorable burial) of residue 64 is the dominant factor impacting stability. The mobility of the axial Met also is proposed to contribute. These results provide a specific illustration of how amino acid side chain mobility and burial or release contribute to protein stability.