Heme-protein interactions in horse heart myoglobin at neutral pH and exposed to acid investigated by time-resolved fluorescence in the pico- to nanosecond time range.
We measured the steady state and time-resolved emission intensity decay of horse heart myoglobin at various pH values from neutral to pH 4.42. The steady state intensity was reversibly increased with the decreasing pH, almost doubling at pH 4.5. Frequency domain data for emission decay were analyzed separately for each pH and simultaneously by global analyses. The results indicated the presence of four lifetime components, conserved throughout the pH titrations at 40, 116, 1363, and 4822 ps, respectively. The titration affected only their fractional intensities. Assignments of the lifetimes were based on the Förster 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 lifetime 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 lifetime was assigned to reversibly heme-dissociated myoglobin. Lorentzian lifetime distributions were narrow for the lifetimes at 40, 116, and 4822 ps, indicating a homogeneous protein structure. Instead the lifetime at 1363 ns had a broad, pH-independent distribution consistent with small angle wobblings of inverted hemes inside the heme pocket. These analyses revealed the presence of three species originating from heme-protein interactions: the native form of crystalline myoglobin, the conformation with disorder hemes, and the reversibly dissociated heme-free myoglobin. There was increased heme inversion and heme dissociability at lower pH, consistent with the titration of the proximal and distal histidines inside the heme pocket.
Gryczynski
Z
Z
Department of Biochemistry, University of Maryland Medical School at Baltimore 21201, USA.
Lubkowski
J
J
Bucci
E
E
D000818:Animals; D006420:Hemeproteins / Q000378:metabolism*; D006736:Horses; D006863:Hydrogen-Ion Concentration; D009211:Myoglobin / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013997:Time Factors; D017186:Titrimetry
D006420:Hemeproteins; D009211:Myoglobin