EPR and ENDOR studies of the distal site coordination of shark heart myoglobin

Abstract

The coordination of small molecules and ions at the sixth position of ferric myoglobins (Mb) from the heart muscle of two species of shark, Lamna ditropis (LD) and Prionace glauca (PG), was studied in frozen solution by electron paramagnetic resonance (EPR) and etectron-nuciear double resonance (ENDOR) spectroscopy. The results were compared with those of horse heart Mb (EQMb). From our preliminary studies of the absorption spectra and partial amino acid sequences of PGMb and LDMb, we expected PGMb to have glutamine (Gln E7) in place of the distal His E7 that is highly conserved in most animals and present in LDMb. Here, we have used proton ENDOR to show that a water molecule coordinates to the ferric ion in both LDMb and PGMb at pH 6.3. The observed proton hyperfine coupling constant of the coordinating water molecule of PGMb is smaller than those of LDMb and EQMb, whieh suggests weaker coordination of the water molecule in PGMb, consistent with the absent distal His (E7). Coordination of several exogenous ligands at the distal site was examined by EPR. PGMb showed a coordination behavior different from that of LDMb and EQMb for 1-methylimidazole, 4-methylimidazole and SCN-, suggesting that its distal site is less sterically hindered. On the basis of the ENDOR and EPR results, we assigned the distal residues of PGMb and LDMb as Gln and His, respectively.

Ultrafast Structural Fluctuations of Myoglobin‐Bound Thiocyanate and Selenocyanate Ions Measured with Two‐Dimensional Infrared Photon Echo Spectroscopy

“…Among the many desirable spectral properties of a good probe, the most noteworthy ones are enhanced dipole strength and long vibrational lifetime. Moreover, the properties of thiocyanate and selenocyanate complexes, which include electronic spin state and binding mode, have been shown to be highly sensitive to the local environment in the heme pocket …”

“…Moreover, the properties of thiocyanate ands elenocyanate complexes, which include electronic spin state and binding mode, have been shown to be highly sensitive to the local environment in the heme pocket. [7] In this work, we presentt he femtosecond 2D-IR and IR pump-probe measurements of the NCs tretcho fM bNCS and MbNCSe complexes. Timescalesa tw hich the instantaneous vibrational frequencies are sampled, are obtained from 2D-IR spectra by analyzing time-dependent lineshape evolutions, from whichh omogeneous and inhomogeneous contributions Structurald ynamics within the distal cavity of myoglobin protein is investigated using 2D-IR and IR pump-probe spectroscopy of the NCs tretchm odes of heme-boundt hiocyanate and selenocyanate ions.…”

“…Interestingly, the ligand‐binding properties have been found to be highly dependent on the local protein environment in the hydrophobic pocket. Properties such as the binding mode, binding affinity, electronic spin state, and induced vibrational and electronic chirality are all determined by the local protein structure. The recent development of ultrafast IR techniques has provided new possibilities for characterizing iron‐bound ligands, which can also serve as vibrational probes, along with the surrounding amino acids that interact directly with the probe under study.…”