TY - JOUR
T1 - 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
AU - Gryczynski, Z.
AU - Lubkowski, J.
AU - Bucci, E.
PY - 1995
Y1 - 1995
N2 - 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 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 lifetime was assigned to Trp-7 with inverted heroes (i.e. rotated 180° around the α-γ-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 heroes 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 disordered 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.
AB - 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 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 lifetime was assigned to Trp-7 with inverted heroes (i.e. rotated 180° around the α-γ-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 heroes 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 disordered 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.
UR - http://www.scopus.com/inward/record.url?scp=0029121701&partnerID=8YFLogxK
U2 - 10.1074/jbc.270.33.19232
DO - 10.1074/jbc.270.33.19232
M3 - Article
C2 - 7642594
AN - SCOPUS:0029121701
SN - 0021-9258
VL - 270
SP - 19232
EP - 19237
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 33
ER -