Folding character of cytochrome c studied by o-nitrobenzyl modification ofmethionine 65 and subsequent ultraviolet light irradiation

The protein folding character of cyt c was studied with the use of a photoc leavable o-nitrobenzyl derivative of Met65 (NBz-Met65). For the NBz-Met65 c yt c, the Sorer absorption band slightly blue shifted compared with the unl abeled cyt c, the 695 nm absorption band related to the Met80 sulfur ligati on to the heme iron disappeared, and its resonance Raman spectrum was chara cteristic of a six-coordinate low-spin species, all characters demonstratin g coordination of a non-native ligand, probably a histidine, instead of Met 80 to the heme iron. The far-UV circular dichroism (CD) spectrum of cyt c w as altered, and the transition midpoint concentration value of guanidine hy drochloride (GdnHCl) for unfolding the protein decreased by 0.9 M by the mo dification, which showed perturbation of the structure and decrease in prot ein stability, respectively. With irradiation of 308 nm laser pulses on the NBz-Met65 cyt c, the Soret absorption band slightly red shifted, the 695 n m absorption band appeared, and the CD spectrum shifted toward that of the native protein, which demonstrated recovery of the methionine heme coordina tion and the native protein structure, due to reconversion of NBz-Met65 to unlabeled methionine. A fast phase was detected as a change in Soret absorb ance with a rate constant of 21 000 +/- 4000 s(-1) during refolding of cyt c initiated by irradiation of a 308 nm pulse on the NBz-MetG5 cyt c in the presence of 2 M GdnHCl. The observed rate constant corresponded well with t hat reported by the tryptophan fluorescence study [Shastry, M. C. R. S., an d Roder, H. (1998) Not. Struct. Biol. 5, 385-392]. The intermediate decayed with a rate constant of 90 +/- 15, followed by another phase with a rate c onstant of 13 +/- 3 s(-1), and was not seen in the absence of GdnHCl.