HEME-PROTEIN INTERACTIONS IN HORSE HEART MYOGLOBIN AT NEUTRAL PH AND EXPOSED TO ACID INVESTIGATED BY TIME-RESOLVED FLUORESCENCE IN THE PICOSECOND TO NANOSECOND TIME RANGE

We measured the steady state and time-resolved emission intensity deca y of horse heart myoglobin at various pH values from neutral to pH 4.4 2. The steady state intensity was reversibly increased with the decrea sing pH, almost doubling at pH 4.5, Frequency domain data for emission decay were analyzed separately for each pH and simultaneously by glob al analyses, The results indicated the presence of four lifetime compo nents, conserved throughout the pH titrations at 40, 116, 1363, and 48 22 ps, respectively, The titration affected only their fractional inte nsitie. Assignments of the lifetimes were based on the Forster theory of radiationless dipole dipole interaction and the atomic coordinates of the system, We assigned the two shorter lifetimes to Trp-14 and Trp -7, respectively, in the presence of normal hemes, The 1363-ps lifetim e was assigned to Trp-7 with inverted hemes (i.e. rotated 180 degrees around the alpha-gamma-meso axis of the porphyrin ring). The 4822-ns l ifetime was assigned to reversibly heme-dissociated myoglobin, Lorentz ian lifetime distributions were narrow for the lifetimes at 40, 116, a nd 4822 ps, indicating a homogeneous protein structure, Instead the li fetime at 1363 ns had a broad, ps-independent distribution consistent with small angle wobblings of inverted hemes inside the heme pocket, T hese analyses revealed the presence of three species originating from heme-protein interactions: the native form of crystalline myoglobin, t he conformation with disordered hemes, and the reversibly dissociated heme-free myoglobin. There was increased heme inversion and heme disso ciability at lower pH, consistent with the titration of the proximal a nd distal histidines inside the heme pocket.