Femtosecond coherence spectroscopy of heme proteins

Femtosecond pump-probe experiments are used to study the photophysics and photochemistry of heme proteins on ultrafast time scales. Using electronic relaxation measurements combined with reaction-driven coherence signals, we determine that, after the photoexcitation of myoglobin (Mb) NO, the bond-breaking time is on the order of the electronic dephasing time (∼ 20 fs) and the electronic ground state of deoxy Mb appears on a time scale of 80 ± 30 fs. This latter time scale is linked to the Fe-His bond compression (T_Fe-His/2 ∼ 75 fs), which is initiated by the photolytic depletion of electron density from the antibonding d_z² orbital. The subsequent depression of the d_x2-y2 orbital energy, and its population as the iron moves out of plane, leads to the formation of the S = 2 electronic ground state and drives the doming of the heme (T_dome/4 ∼ 100 fs). Thus, the initial stage of heme doming is completed in T_Fe-His/2 + T_dome/4 ∼ 180 fs. Samples of hemoglobin (Hb) NO display a strong coherent Fe-His stretching vibration at 230 cm^-1, which appears immediately (∼ 200 fs) after photodissociation. The data also show a lower-frequency component at 95 cm^-1, which we assign to heme doming. The reaction driven coherence of HbNO is observed to be much stronger than the impulsively stimulated Raman coherence of Hb, as might be expected, based on the large relative displacement between reactant and product equilibrium position. The Raman coherence of deoxy Mb is also studied as a function of temperature. As the temperature decreases from 290 to 10 K, the amplitude of the 370 cm^-1 mode increases while the amplitude of the 220 cm^-1 Fe-His stretching mode decreases. Finally, we report preliminary observations of the first Raman coherences in samples of another heme protein, cytochrome P450.