Time-resolved near UV circular dichroism and absorption studies of carbonmonoxymyoglobin photolysis intermediates

Abstract

Abstract Time-resolved circular dichroism (TRCD) spectra of ligand photolysis intermediates of horse skeletal carbonmonoxymyoglobin (MbCO) in the near UV (250–300 run) region are obtained to probe the environment of protein aromatic side chains as a function of time. Since X-ray data reveal compact structures for MbCO and deoxyMb with no apparent pathway for ligand migration through the protein matrix, dynamic protein fluctuations of side chains must be important in facilitating the ligand binding process. Near UV TRCD spectral data are fit to two exponential components with lifetimes of 110±35 μs and 1.5 ± 0.3 ms. Time-resolved absorption spectra in this wavelength region were fit to three lifetimes of 340 ± 100 ns, 830± 240μs and 2.1 ± 0.2 ms. The 830 μs and 2.1 ms processes are associated with bimolecular ligand rebinding and are consistent with lifetimes obtained from spectra in the Soret region of 200 ± 120 ns, 680 ± 80 μs and 1.8 ± 0.05 ms. The absence of the ca. 700μs component in the TRCD data indicates that bimolecular rebinding induces changes in the heme interaction with surrounding polar residues and not with aromatic residues. The 110μs TRCD transient is discussed in terms of motions of tyrosines and/or nearby aromatic groups that are involved in the early stages of ligand rebinding.