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Direct observation of ligand dynamics in cytochrome c.
J Am Chem Soc. 2009 May 06; 131(17):6054-5.JA

Abstract

Horse heart cytochrome c (cyt c) has emerged as a paradigm for the study of protein folding, in large part because the covalently bound heme facilitates its characterization. The folding of reduced cyt c induced by photodissociation of CO from the CO-bound unfolded protein has been extensively studied. Following a nanosecond light pulse, four transitions with time constants of approximately 1-5, 50-100, 200-500, and 1000-10000 micros have been resolved. While originally thought to be associated with CO rebinding to two different partially folded states of cyt c, the two slower processes are now understood to reflect the bimolecular reassociation of CO followed by religation of His18, which by the base elimination mechanism is induced to dissociate after CO photolysis. Thus, it turns out that the two longer time constants do not report on protein folding but instead reflect the complexity of heme ligation. The two shorter time constants have been attributed to ligation at the heme center by Met65 or Met80 and His33 or His26 and have been used to estimate interchain diffusion rates of the protein. Here, to unambiguously determine the post-photodissociation steps involving CO, we have monitored the CO vibration following photodissociation with step-scan FT-IR spectroscopy. We have found that like the longer time scale processes, the 50-100 mus time scale process is associated not with protein dynamics but with CO ligand dynamics. The data clearly demonstrate that whatever the origins of the spectral changes, they clearly involve CO rebinding or changes in the environment of an already bound CO ligand. We speculate that the observed changes reflect His18 religation after fast geminate recombination of the CO. The data suggest that the associated time constant should not be used as a measure of interchain diffusion, and the results emphasize the importance of studying protein folding with probes that have inherently high structural resolution.

Authors+Show Affiliations

Department of Chemistry, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA.No affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, U.S. Gov't, Non-P.H.S.

Language

eng

PubMed ID

19361220

Citation

Thielges, Megan C., et al. "Direct Observation of Ligand Dynamics in Cytochrome C." Journal of the American Chemical Society, vol. 131, no. 17, 2009, pp. 6054-5.
Thielges MC, Zimmermann J, Romesberg FE. Direct observation of ligand dynamics in cytochrome c. J Am Chem Soc. 2009;131(17):6054-5.
Thielges, M. C., Zimmermann, J., & Romesberg, F. E. (2009). Direct observation of ligand dynamics in cytochrome c. Journal of the American Chemical Society, 131(17), 6054-5. https://doi.org/10.1021/ja810155s
Thielges MC, Zimmermann J, Romesberg FE. Direct Observation of Ligand Dynamics in Cytochrome C. J Am Chem Soc. 2009 May 6;131(17):6054-5. PubMed PMID: 19361220.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Direct observation of ligand dynamics in cytochrome c. AU - Thielges,Megan C, AU - Zimmermann,Jörg, AU - Romesberg,Floyd E, PY - 2009/4/14/entrez PY - 2009/4/14/pubmed PY - 2009/7/2/medline SP - 6054 EP - 5 JF - Journal of the American Chemical Society JO - J Am Chem Soc VL - 131 IS - 17 N2 - Horse heart cytochrome c (cyt c) has emerged as a paradigm for the study of protein folding, in large part because the covalently bound heme facilitates its characterization. The folding of reduced cyt c induced by photodissociation of CO from the CO-bound unfolded protein has been extensively studied. Following a nanosecond light pulse, four transitions with time constants of approximately 1-5, 50-100, 200-500, and 1000-10000 micros have been resolved. While originally thought to be associated with CO rebinding to two different partially folded states of cyt c, the two slower processes are now understood to reflect the bimolecular reassociation of CO followed by religation of His18, which by the base elimination mechanism is induced to dissociate after CO photolysis. Thus, it turns out that the two longer time constants do not report on protein folding but instead reflect the complexity of heme ligation. The two shorter time constants have been attributed to ligation at the heme center by Met65 or Met80 and His33 or His26 and have been used to estimate interchain diffusion rates of the protein. Here, to unambiguously determine the post-photodissociation steps involving CO, we have monitored the CO vibration following photodissociation with step-scan FT-IR spectroscopy. We have found that like the longer time scale processes, the 50-100 mus time scale process is associated not with protein dynamics but with CO ligand dynamics. The data clearly demonstrate that whatever the origins of the spectral changes, they clearly involve CO rebinding or changes in the environment of an already bound CO ligand. We speculate that the observed changes reflect His18 religation after fast geminate recombination of the CO. The data suggest that the associated time constant should not be used as a measure of interchain diffusion, and the results emphasize the importance of studying protein folding with probes that have inherently high structural resolution. SN - 1520-5126 UR - https://www.unboundmedicine.com/medline/citation/19361220/Direct_observation_of_ligand_dynamics_in_cytochrome_c_ L2 - https://doi.org/10.1021/ja810155s DB - PRIME DP - Unbound Medicine ER -