HEME PROTEIN DYNAMICS STUDIED BY PHOSPHORESCENCE OF AN EXTERNAL PHOSPHORESCENT PROBE MOLECULE

The rate constant for quenching, k(q), of the phosphorescence of 6-bro mo-2-naphthyl sulfate (BNS) by cytochromes c, cytochrome c peroxidase, catalase, and myoglobin has been measured as a function of temperatur e and solvent viscosity. In aqueous solution at pH 7.0 for cytochromes c and myoglobin the value of k(q) is nearly equal to the rate constan t for diffusional intermolecular contact, which is estimated from the value of k(q) for microperoxidase-11. For cytochrome c peroxidase and catalase K-q is at least 350 times smaller than the rate of diffusiona l quenching, which shows that quenching of BNS phosphorescence occurs predominantly over the short distance between donor and acceptor. The mechanism for cytochrome c and myoglobin is found not to involve stati c quenching, deep penetration of BNS into the globin, or unfolding of the protein to allow contact between hems and BNS. It is concluded tha t quenching occurs by interaction of BNS with the exposed hems edge an d by surface insertion of BNS into the protein to a depth sufficient f or quenching by the unexposed heme. The effect of rapid-diffusional en hancement on k(q) is small. From a comparison of the results for the h ems proteins, a model emerges that describes cytochrome c and myoglobi n as having dynamic surfaces. Sufficient fluctuations persist to allow penetration of polyatomic probe molecules into the protein matrix, bu t the dynamics and/or interior microenvironment acts to increase resis tance with increasing depth of penetration. (C) 1994 Academic Press, I nc.