Equilibrium unfolding of a small bacterial cytochrome, cytochrome c551 from Pseudomonas aeruginosa

The unfolding of the small cytochrome c(551) from the bacterium Pseudomonas aeruginosa has been characterized at equilibrium by circular dichroism (CD) and fluorescence spectroscopy. The process can be described by a two state mechanism and the thermodynamic stability of cytochrome c(551) is found to be smaller than that of the larger horse cytochrome c (Delta G(w)= -8.2 vs. -9.7 kcal/mol); we propose that this finding is related to the absence of an 'omega' loop in the bacterial cytochrome. Cytochrome c(551) loses most of its secondary structure at pH 1.5, The acid transition (pK(A) approximate to 2) is highly cooperative (n greater than or equal to 2); analysis of optical titrations and contact map suggests that (at least) His-16 (proximal Fe3+ Ligand) and Glu-70 are both involved in the acid transition. The role of selected hydrophobic, electrostatic and conformational contributions to the overall stability has been investigated by protein engineering. The equilibrium characterization of wild-type and mutant cytochrome c(551) supports the view that this small cytochrome is an interesting protein to analyze the thermodynamics and the kinetics of folding in comparison with the widely studied horse cytochrome c. (C) 1998 Federation of European Biochemical Societies.