Proximal and distal influences on ligand binding kinetics in microperoxidase and heme model compounds.

Abstract

We use laser flash photolysis and time-resolved Raman spectroscopy of CO-bound heme complexes to study proximal and distal influences on ligand rebinding kinetics. We report kinetics of CO rebinding to microperoxidase (MP) and 2-methylimidazole ligated Fe protoporphyrin IX in the 10 ns to 10 ms time window. We also report CO rebinding kinetics of MP in the 150 fs to 140 ps time window. For dilute, micelle-encapsulated (monodisperse) samples of MP, we do not observe the large amplitude geminate decay at approximately 100 ps previously reported in time-resolved IR measurements on highly concentrated samples [Lim, M., Jackson, T. A., and Anfinrud, P. A. (1997) J. Biol. Inorg. Chem. 2, 531-536]. However, for high concentration aggregated samples, we do observe the large amplitude picosecond CO geminate rebinding and find that it is correlated with the absence of the iron-histidine vibrational mode in the time-resolved Raman spectrum. On the basis of these results, the energetic significance of a putative distal pocket CO docking site proposed by Lim et al. may need to be reconsidered. Finally, when high concentration samples of native myoglobin (Mb) were studied as a control, an analogous increase in the geminate rebinding kinetics was not observed. This verifies that studies of Mb under dilute conditions are applicable to the more concentrated regime found in the cellular milieu.