Conformational Relaxation in Hemoproteins:  The Cytochrome P-450_camCase

Abstract

Photodissociation of (CO)P-450(cam)(substrate) complexes was found to trigger a conformational relaxation process that interferes with ligand rebinding at temperatures as low as 140 K even though the protein conformational substates (CS(1)) remain frozen. To analyze the rebinding and relaxation kinetics, we developed a model that takes the distribution of relaxation rates explicitly into account and in which rebinding and relaxation rates are connected by a linear free energy relation. In all complexes heme relaxation occurs first and is probably faster than 100 ns even at 77 K. This is the only process found in substrate-free P-450(cam). Above 140 K and in the presence of a substrate, this initial, fast rebinding state (P) progressively relaxes to another state (P degrees ) in which rebinding is slower. The relaxation rate is independent of solvent rigidity and is governed by the protein's internal dynamics. Rebinding enthalpies in P and P degrees as well as the enthalpy shift brought about by relaxation correlate with the substrate propensity to block access to the iron site. In P degrees the barrier is higher because the substrate is closer to the heme normal and exerts more steric repulsion for CO binding. The relaxation process implies the return of substrate and heme to their ligand-free positions in which access to the heme is reduced.

Competition with Xenon Elicits Ligand Migration and Escape Pathways in Myoglobin

“…In P450 cam the G II process appears at much lower temperature (T 140 K) and is presumed to correspond to a rearrangement of the distal pocket, as soon as the protein interior recovers sufficient local mobility for the heme and substrate to ''relax'' to the more crowded, deliganded conformation. Such local motions being less dependent on the fluctuations of the protein matrix, the change in the kinetic regime was observed well below T g and the kinetic ''relaxation'' rate was shown to be decoupled from the solvent's dielectric relaxation (Tetreau et al, 2000).…”