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Zinc-porphyrin Solvation in Folded and Unfolded States of Zn-cytochrome c

Kim, Judy E. and Pribisko, Melanie A. and Gray, Harry B. and Winkler, Jay R. (2004) Zinc-porphyrin Solvation in Folded and Unfolded States of Zn-cytochrome c. Inorganic Chemistry, 43 (25). pp. 7953-7960. ISSN 0020-1669. doi:10.1021/ic048972l.

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After a brief review of the use of photochemical triggers and heme metal substitution to probe the folding dynamics of cytochrome c, we present new results on the photophysics and photochemistry of folded and unfolded states of the zinc-substituted protein (Zn-cyt c). Our measurements of Zn-cyt c triplet state decay kinetics reveal a systematic isotope effect on lifetimes:  the decay in the folded protein (τ_H_2_O ∼ 10 ms) is only modestly affected by isotopically substituted buffers (k_H_2_O/k_D_2_O = 1.2), whereas a reduced triplet lifetime (∼1.3 ms) and greater isotope effect (1.4) were found for the chemically denatured, fully unfolded protein. The shortest lifetime (0.1−0.4 ms) and greatest isotope effect (1.5) were found for a fully exposed model compound, zinc-substituted N-acetyl-microperoxidase-8 (ZnAcMP8), implying that the unfolded protein provides some protection to the Zn-porphyrin group even under fully denaturing conditions. Further evidence for partial structure in unfolded Zn-cyt c comes from bimolecular quenching experiments using Ru(NH_3)_6^(3+) as an external Zn-porphyrin triplet state quencher. In the presence of quencher, partially unfolded protein at midpoint guanidinium chloride (GdmCl) and urea concentrations exhibits biphasic triplet decay kinetics, a fast component corresponding to an extended, solvent-exposed state (6.6 × 10^8 M^(-1) s^(-1) in GdmCl, 6.3 × 10^8 M^(-1) s^(-1) in urea) and a slow component attributable to a compact, relatively solvent-inaccessible, state (5.9 × 10^7 M^(-1) s^(-1) in GdmCl, 8.6 × 10^6 M^(-1) s^(-1) in urea). The variation in Zn-porphyrin solvation for the compact states in the two denaturants reveals that the cofactor in the partially unfolded protein is better protected in urea solutions.

Item Type:Article
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Gray, Harry B.0000-0002-7937-7876
Winkler, Jay R.0000-0002-4453-9716
Additional Information:© 2004 American Chemical Society. Received 29 July 2004. Published online 5 November 2004. Published in print 1 December 2004. We thank Kate Pletneva for assistance with zinc incorporation into cytochrome c, and Jennifer Lee and John Magyar for helpful discussions. J.E.K. is supported by a Ruth L. Kirschstein National Research Service Award from the National Institutes of Health. This research was supported by the National Institutes of Health (GM068461) and the Arnold and Mabel Beckman Foundation.
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Arnold and Mabel Beckman FoundationUNSPECIFIED
NIH Predoctoral FellowshipUNSPECIFIED
Issue or Number:25
Record Number:CaltechAUTHORS:20170425-074119551
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Official Citation:Zinc-porphyrin Solvation in Folded and Unfolded States of Zn-cytochrome c Judy E. Kim, Melanie A. Pribisko, Harry B. Gray, and Jay R. Winkler Inorganic Chemistry 2004 43 (25), 7953-7960 DOI: 10.1021/ic048972l
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:76896
Deposited By: Ruth Sustaita
Deposited On:25 Apr 2017 14:59
Last Modified:15 Nov 2021 17:03

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