Time-dependent fifth-order bands in nominally third-order 2D IR vibrational echo spectra

Progress in the field of 2D IR vibrational spectroscopy has been bolstered by the production of intense mid-IR laser pulses. As higher-energy pulses are employed, a concomitant increase occurs in the likelihood of fifth-order contributions to the 2D IR spectra. We report the appearance of fifth-order signals in 2D IR spectra of CO bound to the active site of the enzyme cytochrome P450(cam) with the substrate norcamphor. Two bands with novel time dependences, one on the diagonal and one off-diagonal, are not accounted for by normal third-order interactions. These bands are associated with a ν = 1-2 vibrational transition frequency. Both bands decay to 0 and then grow back in with opposite sign. The diagonal band is positive at short time, decays to 0, reappears with negative sign, before eventually decaying to 0. The off-diagonal band is negative at short time, decays to 0, reappears positive, and then decays to 0. The appearance and time dependence of these bands are characterized. Understanding these fifth-order bands is useful because they may be misidentified with time-dependent bands that arise from other processes, such as chemical exchange, vibrational coupling, or energy transfer. The presence and unusual time dependences of the fifth-order bands are reproduced with model calculations that account for the fact that vibrational relaxation from the ν = 2 to 1 level is approximately a factor of 2 faster than that from the ν = 1 to 0 level.