Differences in thermal stability between reduced and oxidized cytochrome b562 from Escherichia coli.
The thermal stabilities of ferri- and ferrocytochrome b562 were examined. Thermally induced spectral changes, monitored by absorption and second-derivative spectroscopies, followed the dissociation of the heme moiety and the increased solvation of tyrosine residue(s) located in close proximity to the heme binding site. All observed thermal transitions were independent of the rate of temperature increase (0.5-2 degrees C/min), and the denatured protein exhibited partial to near-complete reversibility upon return to ambient temperature. The extent of renaturation of cytochrome b562 is dependent on the amount of time the unfolded conformer is exposed to temperatures above the transition temperature, Tm. All thermally induced spectra changes fit a simple two-state model, and the thermal transition was assumed to be reversible. The thermal transition for ferrocytochrome b562 yielded Tm and van't Hoff enthalpy (delta HvH) values of 81.0 degrees C and 137 kcal/mol, respectively. In contrast, Tm and delta HvH values obtained for the ferricytochrome were 66.7 degrees C and 110 kcal/mol, respectively. The estimated increase in the stabilization free energy at the Tm of ferricytochrome b562 following the one-electron reduction to the ferrous form, where delta delta G = delta Tm delta Sm [delta Sm = 324 cal/(K.mol), delta Tm = 14.3 degrees C] [Becktel, W. J., & Schellman, J. A. (1987) Biopolymers 26, 1859-1877], is 4.6 kcal/mol.
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Laboratory of Biochemistry, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892.
D003573:Cytochrome b Group / Q000737:chemistry*; D004579:Electron Transport; D004795:Enzyme Stability / Q000187:drug effects; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D006418:Heme / Q000737:chemistry; D007700:Kinetics; D010084:Oxidation-Reduction; D011487:Protein Conformation; D011489:Protein Denaturation; D013056:Spectrophotometry, Ultraviolet; D013816:Thermodynamics
D003573:Cytochrome b Group; D029968:Escherichia coli Proteins; D006146:Guanidines; D006418:Heme; C017850:cytochrome b562, E coli; D019791:Guanidine