Ligand binding to heme proteins is studied by using flash photolysis over wide ranges in time (100 ns-1 ks) and temperature (10-320 K). Below about 200 K in 75% glycerol/water solvent, ligand rebinding occurs from the heme pocket and is nonexponential in time. The kinetics is explained by a distribution, g(H), of the enthalpic barrier of height H between the pocket and the bound state. Above 170 K rebinding slows markedly. Previously we interpreted the slowing as a "matrix process" resulting from the ligand entering the protein matrix before rebinding. Experiments on band III, an inhomogeneously broadened charge-transfer band near 760 nm (approximately 13,000 cm-1) in the photolyzed state (Mb*) of (carbonmonoxy)myoglobin (MbCO), force us to reinterpret the data. Kinetic hole-burning measurements on band III in Mb* establish a relation between the position of a homogeneous component of band III and the barrier H. Since band III is red-shifted by 116 cm-1 in Mb* compared with Mb, the relation implies that the barrier in relaxed Mb is 12 kJ/mol higher than in Mb*. The slowing of the rebinding kinetics above 170 K hence is caused by the relaxation Mb*----Mb, as suggested by Agmon and Hopfield [(1983) J. Chem. Phys. 79, 2042-2053]. This conclusion is supported by a fit to the rebinding data between 160 and 290 K which indicates that the entire distribution g(H) shifts. Above about 200 K, equilibrium fluctuations among conformational substates open pathways for the ligands through the protein matrix and also narrow the rate distribution. The protein relaxations and fluctuations are nonexponential in time and non-Arrhenius in temperature, suggesting a collective nature for these protein motions. The relaxation Mb*----Mb is essentially independent of the solvent viscosity, implying that this motion involves internal parts of the protein. The protein fluctuations responsible for the opening of the pathways, however, depend strongly on the solvent viscosity, suggesting that a large part of the protein participates. While the detailed studies concern MbCO, similar data have been obtained for MbO2 and CO binding to the beta chains of human hemoglobin and hemoglobin Zürich. The results show that protein dynamics is essential for protein function and that the association coefficient for binding from the solvent at physiological temperatures in all these heme proteins is governed by the barrier at the heme.

Initiation of protein folding by light can dramaticafly improve the time resolution of kinetic studies. Here we present an example ofan optically triggered folding reaction by using nanosecond photodissociation of the heme-carbon monoxide complex of reduced cytochrome c. The optical trigger is based on the observation that under destabilizing conditions cytochrome c can be unfolded by preferential binding of carbon monoxide to the covalently attached heme group in the unfolded state. Photodissociation of the carbon monoxide thus triggers the folding reaction. We used time-resolved absorption spectroscopy to monitor binding at the heme. Before folding begins we observe transient binding of both nonnative and native ligands from the unfolded polypeptide on a microsecond time scale. Kinetic modeling suggests that the intramolecular binding of methionine-65 and -80 is faster than that of histidine-26 and -33, even though the histidines are doser to the heme. This optical trigger should provide a powerful method for studying chain collapse and secondary structure formation in cytochrome c without any limitations in time resolution.Protein folding generally occurs in two phases, one rapid and one slow. The rapid phase is the collapse of the unfolded polypeptide into a compact structure and the formation of secondary structure. The slow phase is the subsequent, often multistep rearrangement to the native conformation (1-7). Because of the limited time resolution (milliseconds) of conventional stopped-flow mixing experiments, the complete time course of protein folding has not yet been observed. A dramatic improvement in time resolution would result if protein folding could be initiated by light.Here we present an example of an optically triggered folding reaction. We take advantage of the fact that under destabilizing conditions cytochrome c can be unfolded by preferential binding of carbon monoxide (CO) to the covalently attached heme group in the unfolded state. The folding reaction can thus be triggered by photodissociating the CO complex. In this study, we used time-resolved absorption spectroscopy with nanosecond lasers to monitor binding events at the heme (8-11). Although rebinding of CO prevents the complete formation of the native conformation, the rapid recovery of the sample permits repetitive photolysis and therefore the acquisition of high signal/noise transient spectra for investigating submillisecond events. Simulations of the multiwavelength data with kinetic models were carried out to generate the spectra of intermediates, as well as the rate constants connecting them. We find that before folding begins there is transient binding of both nonnative and native ligands from the unfolded polypeptide chain on a microsecond time scale. MATERIALS AND METHODSTye VI horse heart cytochrome c from Sigma was purified by ion-exchange chromatography using carboxymethyl-cellulose (Whatman) (12), reduced with sodium dithionite anaerobically, and separated anaerobically from the sodium dithionite by gel filtrat...

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