Protein dynamics Comparative investigation on heme-proteins with different physiological roles Ernesto E. Di lorio,* Urs R. Hiltpold,* Dorde Filipovic,* Kaspar H. Winterhalter,* Enrico Gratton,* Eugenio Vitrano,§ Antonio Cupane,§ Maurizio Leone,§ and Lorenzo Cordone§ *Laboratorium fur Biochemie I, Eidgenossische Technische Hochschule 8092 Zurich, Switzerland; *Department of Physics, University of Illinois at Urbana-Champaign, Urbana, IL 61801 USA; and 'Istituto di Fisica and GNSM-CISM, 90123, Palermo, Italy ABSTRACT We report the low temperature carbon monoxide recombination kinetics after photolysis and the temperature dependence of the visible absorption spectra of the isolated aSH_CO and 13SH-CO subunits from human hemoglobin A in ethylene glycol/water and in glycerol/water mixtures. Kinetic measurements on sperm whale (Physeter catodon) myoglobin and previously published optical spectroscopy data on the latter protein and on human hemoglobin A, in both solvents, (Cordone, L., A. Cupane, M. Leone, E. Vitrano, and D. Bulone. 1988. J. Mol. Biol. 199:312-218) are taken as reference. Low temperature flash photolysis data are analyzed within the multiple substates model proposed by Frauenfelder and co-workers (Austin, R. H., K. W. Beeson, L. Eisenstein, H. Frauenfelder, and 1. C. Gunsalus. 1975. Biochemistry. 14:5355-5373). Within this model a distribution of activation enthalpies for ligand binding accounts for the structural heterogeneity of the protein, while the preexponential factor, containing also the entropic contribution to the free energy of the process, is considered to be constant for all conformational substates. Optical spectra are deconvoluted in gaussian components and the temperature dependence of the moments of the resulting bands is analyzed, within the harmonic Frank-Condon approximation, to obtain information on the stereodynamic properties of the heme pocket. The kinetic and spectral parameters thus obtained are found to be protein dependent also with respect to their sensitivity to changes in the composition of the external medium. A close correlation between the kinetic and spectral features is observed for the proteins examined under all experimental conditions studied. The results reported are discussed in terms of differences in the heme pocket structure and in the conformational heterogeneity among the various proteins, as related to their different capability to accommodate constraints imposed by the external medium. INTRODUCTION It is generally accepted that structural fluctuations are necessary for the penetration of ligands or substrates from the solvent to the active site, through the protein matrix. However, a regulatory role of protein dynamics at the functional level is still often questioned. To gain insights into this matter, we started a comparative study on the dynamic properties of proteins with different physiological roles, i.e., myoglobins on one side and hemoglobin or its isolated subunits on the other, using optical spectroscopy and flash photolysis as experimen- tal approaches. The temperature dependence of optical absorption spectra of heme proteins can be related to the interac- tions of the optical electrons with nuclear vibrations (Cordone et al., 1986, 1988; Cupane et al., 1988; Leone et al., 1987; Schomacker and Champion, 1986; Srajer et al., 1986). Based on this correlation, the optical spectra thermal behavior of SW-Mb' and Hb-A has been investi- This work is dedicated, with immense admiration and gratitude, to Prof. Jeffries Wyman in occasion of his 90th birthdate. Dr. Dorde Filipovic's present address in Department of Biochemistry, University of Illinois at Urbana Champaign, 1209 West California Street, Urbana, IL 61801 USA. gated (Cordone et al., 1986, 1988; Cupane et al., 1988; Leone et al., 1987). The temperature dependence (be- tween 20 and 300 K) of the first (MJ) and second (M2) moment of all observed optical absorption bands of human deoxy, oxy-, and carbonyl-HbA, can be rational- ized in terms of a harmonic model for the coupling between the optical electrons and the nearby nuclei (Eq. 2 in Theoretical Background) and is not influenced by the replacement of ethylene glycol with glycerol in the external medium. In contrast, in deoxy, oxy-, and car- bonyl-SW-Mb a considerable solvent dependence of M, is observed, but not of M2, and the model fails in predicting the thermal evolution of the first moment while being successful with the second. The different behavior of SW-Mb as compared with Hb was suggested to arise from the different aggregation state of the two 'Abbreviations used in this paper: Hb = hemoglobin; HbA = major human adult hemoglobin; SW-Mb = sperm whale (Physeter catodon) myoglobin; PMB = p-mercuribenzoate; aSH and 3H = isolated a and f subunits from HbA with free SH groups; a"MB and 3PMB = isolated a and 1B subunits from HbA with the SH groups reacted with PMB; MO, M,, and M2 = zeroth, first and second moments of deconvoluted optical absorption bands related, respectively, to their area, peak position and width. 74 00639/1/3721 $20 ipy.J ipyia Biophys. J. 5 Biophysical So7iety Volume 59 March 1991 742-754 742 0006-3495/91/03/742/13 $2.00