Dynamics of Geminate Rebinding of CO to Cytochrome c in Guanidine HCl Probed by Femtosecond Vibrational Spectroscopy

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• 作者：Jooyoung Kim ; Jaeheung Park ; Taegon Lee ; Youngshang Pak ; Manho Lim
• 刊名：The Journal of Physical Chemistry B
• 出版年：2013
• 出版时间：May 2, 2013
• 年：2013
• 卷：117
• 期：17
• 页码：4934-4944
• 全文大小：438K
• 年卷期：v.117,no.17(May 2, 2013)
• ISSN：1520-5207

文摘

Femtosecond vibrational spectroscopy was used to probe the rebinding dynamics of CO to cytochrome c (Cytc) in 1.8 and 7 M guanidine HCl (GdnHCl) after photodeligation of the corresponding CO-bound protein in D₂O buffer (pD = 7.4) at 283 K. Geminate rebinding (GR) dynamics of CO to the folded Cytc in 1.8 M GdnHCl (nCytc) is similar to that to chemically modified cytochrome c (cCytc), suggesting that the overall conformations of nCytcCO and cCytcCO are similar. About 86% of the dissociated CO molecules were geminately rebound to nCytc nonexponentially within 1 ns. The efficient GR of CO to the folded Cytc can be attributed to the organized protein matrix near the active site of nCytc that provides an efficient trap for the diffusing CO ligand after photodissociation. Although the concentration of nCytc did not affect its GR yield of CO, GR yield of CO to the unfolded Cytc in 7 M GdnHCl (uCytc) increased from 5 to 30% as the protein concentration increased from 0.3 to 9 mM. Time-resolved spectra of the ¹³CO dissociated from both 9 mM nCytc¹³CO and 9 mM uCytc¹³CO showed a growing band with a peak at 2090 cm^鈥? on the picosecond time scale, which was assigned to ¹³CO in D₂O solvent. At 1 ns, the fraction of the CO band in the solvent was about 10% of the nascent photodeligated protein in nCytc and more than 50% in the concentrated uCytc. Whereas a small opening in the active site of nCytc is responsible for the ultrafast escape of CO to solution in the folded protein, a large fraction of the CO escape to the solvent in uCytc results from the denatured structure of the active site in the unfolded protein. The spectrum of the CO dissociated from the concentrated uCytcCO contained a band that decayed as efficiently as that for the folded protein, suggesting that some fraction of uCytcCO might form aggregates even in 7 M denaturant, such that the aggregate acts as an efficient trap for the diffusing CO after deligation. No hint of precipitate in the concentrated uCytcCO and protein refolding upon dilution of the GdnHCl indicate that the aggregate does not grow continuously but remains as a soluble oligomer. The delayed appearance of the solvated CO and the inefficient GR of CO in uCytcCO suggest that the monomeric unfolded CytcCO so loosely arranged that the protein matrix cannot trap CO efficiently but the bound CO is still buried within hydrophobic residues even under the harsh denaturation condition.