Photoexcitation Dynamics of NO-Bound Ferric Myoglobin Investigated by Femtosecond Vibrational Spectroscopy

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• 作者：Jaeheung Park ; Taegon Lee ; Jaehun Park ; Manho Lim
• 刊名：The Journal of Physical Chemistry B
• 出版年：2013
• 出版时间：March 14, 2013
• 年：2013
• 卷：117
• 期：10
• 页码：2850-2863
• 全文大小：556K
• 年卷期：v.117,no.10(March 14, 2013)
• ISSN：1520-5207

文摘

Femtosecond vibrational spectroscopy was used to investigate the photoexcitation dynamics of NO-bound ferric myoglobin (Mb^IIINO) in D₂O solution at 294 K after excitation with a 575 nm pulse. The stretching mode of NO in Mb^IIINO consists of a major band at 1922 cm^鈥? (97.7%) and a minor band at 1902 cm^鈥? (2.3%), suggesting that Mb^IIINO in room temperature solution has two conformational substates. The time-resolved spectra show small but significant new absorption features at the lower-energy side of the main band (1920鈥?800 cm^鈥?). One new absorption feature in the region of 1920鈥?880 cm^鈥? exhibits the ¹⁵NO isotope shift (37 cm^鈥?) the same as that of the NO band in the ground electronic state of Mb^IIINO. This absorption shifts toward higher energy and narrows with a time constant of 2.4 ps, indicating that it evolves with rapid electronic and thermal relaxation of the photoexcited Mb^IIINO without photodeligation of the NO from the heme. Absorption features assigned to proteins undergoing thermal relaxation without NO deligation add up to 14 卤 1% of the total bleach, implying that the photolysis quantum yield of Mb^IIINO with a Q-band excitation is 鈮?.86 卤 0.01. The remaining absorption bands peaked near 1867, 1845, and 1815 cm^鈥?, each showing the ¹⁵NO isotope shift the same as that of the free NO radical (33 cm^鈥?), were assigned to the vibrational band of the photodeligated NO, the NO band of Mb^IIINO in an intermediate electronic state with low-spin Fe(III)鈥揘O(radical) character (denoted as the R state), and the NO band of the vibrationally excited NO in the R state, respectively. A kinetics model successfully reproducing the time-dependent intensity changes of the transient bands suggests that every rebound NO forms the R state that eventually relaxes into the ground electronic state nonexponentially. Most of the photodissociated NO undergoes fast geminate recombination (GR), and the rebinding kinetics depends on the conformation of the protein. GR of NO to Mb^III in the major conformation shows highly nonexponential kinetics described by a stretched exponential function, exp(鈭?t/290 ps)^0.44. The NO rebinding to Mb^III in the minor conformation is exponential, exp(鈭?i>t/1.8 ns), suggesting that the distal histidine, the interaction of which dictates the conformation of Mb^IIINO, participates in mediating the binding of NO to Mb^III. In Mb^IIINO, the elusive low-spin Fe(III)鈥揘O(radical) state, proposed in electronic structure calculations, indeed exists at >12 kJ/mol above the ground state and takes part in the bond formation of Fe(III)鈥揘O, suggesting that it plays a significant role in the function of NO-bound ferric protein. Time-resolved vibrational spectra with high sensitivity reveal rich photophysical and photochemical processes of photoexcited Mb^IIINO.