| Abstract
| - Spectrally resolved infrared stimulated vibrational echo experiments are used to measure the vibrationaldephasing of a CO ligand bound to the heme cofactor in two mutated forms of the cytochrome c552 fromHydrogenobacter thermophilus. The first mutant (Ht-M61A) is characterized by a single mutation of Met61to an Ala (Ht-M61A), while the second variant is doubly modified to have Gln64 replaced by an Asn inaddition to the M61A mutation (Ht-M61A/Q64N). Multidimensional NMR experiments determined that thegeometry of residue 64 in the two mutants is consistent with a non-hydrogen-bonding and hydrogen-bondinginteraction with the CO ligand for Ht-M61A and Ht-M61A/Q64N, respectively. The vibrational echoexperiments reveal that the shortest time scale vibrational dephasing of the CO is faster in the Ht-M61A/Q64N mutant than that in Ht-M61A. Longer time scale dynamics, measured as spectral diffusion, are unchangedby the Q64N modification. Frequency−frequency correlation functions (FFCFs) of the CO are extracted fromthe vibrational echo data to confirm that the dynamical difference induced by the Q64N mutation is primarilyan increase in the fast (hundreds of femtoseconds) frequency fluctuations, while the slower (tens of picoseconds)dynamics are nearly unaffected. We conclude that the faster dynamics in Ht-M61A/Q64N are due to thelocation of Asn64, which is a hydrogen bond donor, above the heme-bound CO. A similar difference in COligand dynamics has been observed in the comparison of the CO derivative of myoglobin (MbCO) and itsH64V variant, which is caused by the difference in axial residue interactions with the CO ligand. The resultssuggest a general trend for rapid ligand vibrational dynamics in the presence of a hydrogen bond donor.
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