Folding character of cytochrome c studied by o-nitrobenzyl modification of methionine 65 and subsequent ultraviolet light irradiation.
The protein folding character of cyt c was studied with the use of a photocleavable o-nitrobenzyl derivative of Met65 (NBz-Met65). For the NBz-Met65 cyt c, the Soret absorption band slightly blue shifted compared with the unlabeled cyt c, the 695 nm absorption band related to the Met80 sulfur ligation to the heme iron disappeared, and its resonance Raman spectrum was characteristic of a six-coordinate low-spin species, all characters demonstrating coordination of a non-native ligand, probably a histidine, instead of Met80 to the heme iron. The far-UV circular dichroism (CD) spectrum of cyt c was altered, and the transition midpoint concentration value of guanidine hydrochloride (GdnHCl) for unfolding the protein decreased by 0.9 M by the modification, which showed perturbation of the structure and decrease in protein stability, respectively. With irradiation of 308 nm laser pulses on the NBz-Met65 cyt c, the Soret absorption band slightly red shifted, the 695 nm absorption band appeared, and the CD spectrum shifted toward that of the native protein, which demonstrated recovery of the methionine heme coordination and the native protein structure, due to reconversion of NBz-Met65 to unlabeled methionine. A fast phase was detected as a change in Soret absorbance 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-Met65 cyt c in the presence of 2 M GdnHCl. The observed rate constant corresponded well with that reported by the tryptophan fluorescence study [Shastry, M. C. R. S., and Roder, H. (1998) Nat. Struct. Biol. 5, 385-392]. The intermediate decayed with a rate constant of 90 +/- 15, followed by another phase with a rate constant of 13 +/- 3 s(-)(1), and was not seen in the absence of GdnHCl.
Okuno
T
T
Department of Chemistry, Graduate School of Science, Nagoya University, Japan.
Hirota
S
S
Yamauchi
O
O
D000818:Animals; D002942:Circular Dichroism; D003574:Cytochrome c Group / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D004795:Enzyme Stability; D019791:Guanidine; D006736:Horses; D008715:Methionine / Q000737:chemistry*; D009206:Myocardium / Q000201:enzymology; D009578:Nitrobenzenes / Q000737:chemistry*; D011489:Protein Denaturation; D017510:Protein Folding; D014466:Ultraviolet Rays
D003574:Cytochrome c Group; D009578:Nitrobenzenes; D008715:Methionine; C036077:nitrobenzene; D019791:Guanidine