文摘
Femtosecond vibrational spectroscopy was used to investigate the photoexcitation dynamics of NO-bound ferric myoglobin (MbIIINO) in D2O solution at 294 K after excitation with a 575 nm pulse. The stretching mode of NO in MbIIINO consists of a major band at 1922 cm鈥? (97.7%) and a minor band at 1902 cm鈥? (2.3%), suggesting that MbIIINO 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 MbIIINO. 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 MbIIINO 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 MbIIINO 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 MbIIINO 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 MbIII 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 MbIII 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 MbIIINO, participates in mediating the binding of NO to MbIII. In MbIIINO, 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 MbIIINO.