Spectrally resolved three-pulse stimulated vibrational echo experiments are used as the basis for structural assignments of the A 1 and A 3 spectroscopic substates in the IR spectrum of the carbon monoxide (CO) stretch of carbonmonoxymyoglobin (MbCO). The measured dephasing dynamics of these substates is compared to the dephasing dynamics of MbCO predicted from molecular dynamics (MD) simulations. We assign the A 1 and A 3 substates to different protein conformations on the basis of the agreement between the measured and computed vibrational echoes. In the A 1 substate, the N -H proton and N δ of His64 are equidistant from the ligand, whereas in the A 3 substate, the N -H of His64 is oriented toward the CO.Structural information is essential for understanding the functions of proteins. Techniques such as X-ray diffraction, 1-3 neutron diffraction, 4 and 2D-NMR 5 have been used to investigate the structures of many proteins and a wide variety of other biomolecules. Despite the power of these methods, the identification of conformational substates that interconvert rapidly is difficult because of the limited time resolution of these techniques. A long-standing problem of this type is the assignment of the A conformational substates of the protein carbonmonoxymyoglobin (MbCO).The infrared (IR) spectrum of the CO stretching mode of MbCO has three absorption bands, denoted A 0 (∼1965 cm -1 ), A 1 (∼1944 cm -1 ), and A 3 (∼1930 cm -1 ), as shown in Figure 1. 6-8 It has been suggested that different electrostatic environments in the heme pocket arising from distinct structures are largely responsible for the observed bands. 9 The distal histidine His64 plays a prominent role in determining the CO stretching frequencies, but the tautomerization and orientation of this residue remain controversial. [1][2][3]10,11 His64 has two titratable nitrogens, N δ and N , either of which can be oriented toward the ligand through rotation of the imidazole ring. This residue is also fairly mobile and has been observed at a wide variety of distances from the CO ligand. 2,3,12 At low pH, His64 is thought to be doubly protonated and has been observed in the low pH crystal structure rotated out of the heme binding pocket away from the CO ligand. 12 This conformer, with little interaction between His64 and the ligand, is thought to correspond to the A 0 substate, because at low pH the A 0 line is the most intense IR absorption band. In addition, mutations of His64 to apolar residues produce an A substate band at approximately the same frequency as the A 0 line. 9,13 At pH greater than 6, the A 1 and A 3 substates are the most populated. 7,14 Crystal structures at neutral pH indicate that His64 is rotated into the heme pocket and is much closer to the ligand than in the A 0 substate, but the exact orientation of His64 is uncertain. 2,3 Also, the tautomerization state of the singly protonated His64 at neutral pH is unclear.Structural calculations have provided insight into the origins of the A 1 and A 3 substates. Rovira et al. 10 performed...

Ultrafast spectroscopy is dominated by time domain methods such as pump-probe and, more recently, 2D-IR spectroscopies. In this paper, we demonstrate that a mixed frequency/time domain ultrafast four wave mixing (FWM) approach not only provides similar capabilities, but it also provides optical analogues of multiple- and zero-quantum heteronuclear nuclear magnetic resonance (NMR). The method requires phase coherence between the excitation pulses only over the dephasing time of the coherences. It uses twelve coherence pathways that include four with populations, four with zero-quantum coherences, and four with double-quantum coherences. Each pathway provides different capabilities. The population pathways correspond to those of two-dimensional (2D) time domain spectroscopies, while the double- and zero-quantum coherence pathways access the coherent dynamics of coupled quantum states. The three spectral and two temporal dimensions enable the isolation and characterization of the spectral correlations between different vibrational and/or electronic states, coherence and population relaxation rates, and coupling strengths. Quantum-level interference between the direct and free-induction decay components gives a spectral resolution that exceeds that of the excitation pulses. Appropriate parameter choices allow isolation of individual coherence pathways. The mixed frequency/time domain approach allows one to access any set of quantum states with coherent multidimensional spectroscopy.

2D spectrally resolved ultrafast (<200 fs) IR vibrational echo experiments were performed on Rh(CO)(2)acac [(acetylacetonato)dicarbonylrhodium (I)]. The 2D spectra display features that reflect the 0-1 and 1-2 transitions and the combination band transition of the symmetric (S) and antisymmetric (A) CO stretching modes. Three oscillations in the data arise from the frequency difference between the S and A modes (quantum beats) and the S and A anharmonicities. A new explanation is given for these "anharmonic" oscillations. Calculations show that spectral resolution enables the 0-1 and 1-2 dephasing to be measured independently.