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Primary protein response after ligand photodissociation in carbonmonoxy myoglobin.

ABSTRACT: Time-resolved UV resonance Raman (UVRR) spectroscopic studies of WT and mutant myoglobin were performed to reveal the dynamics of protein motion after ligand dissociation. After dissociation of carbon monoxide (CO) from the heme, UVRR bands of Tyr showed a decrease in intensity with a time constant of 2 ps. The intensity decrease was followed by intensity recovery with a time constant of 8 ps. On the other hand, UVRR bands of Trp residues located in the A helix showed an intensity decrease that was completed within the instrument response time. The intensity decrease was followed by an intensity recovery with a time constant of approximately 50 ps and lasted up to 1 ns. The time-resolved UVRR study of the myoglobin mutants demonstrated that the hydrophobicity of environments around Trp-14 decreased, whereas that around Trp-7 barely changed in the primary protein response. The present data indicate that displacement of the E helix toward the heme occurs within the instrument response time and that movement of the FG corner takes place with a time constant of 2 ps. The finding that the instantaneous motion of the E helix strongly suggests a mechanism in which protein structural changes are propagated from the heme to the A helix through the E helix motion.

SUBMITTER: Sato A

PROVIDER: S-EPMC1887578 | biostudies-literature | 2007 Jun

REPOSITORIES: biostudies-literature

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Primary protein response after ligand photodissociation in carbonmonoxy myoglobin.

Sato Akira A Gao Ying Y Kitagawa Teizo T Mizutani Yasuhisa Y

Proceedings of the National Academy of Sciences of the United States of America 20070521 23

Time-resolved UV resonance Raman (UVRR) spectroscopic studies of WT and mutant myoglobin were performed to reveal the dynamics of protein motion after ligand dissociation. After dissociation of carbon monoxide (CO) from the heme, UVRR bands of Tyr showed a decrease in intensity with a time constant of 2 ps. The intensity decrease was followed by intensity recovery with a time constant of 8 ps. On the other hand, UVRR bands of Trp residues located in the A helix showed an intensity decrease that ...[more]

PMID: 17517618

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Project description:Molecular dynamics simulations of the photodissociated state of carbonmonoxy myoglobin (MbCO) are presented using a fluctuating charge model for CO. A new three-point charge model is fitted to high-level ab initio calculations of the dipole and quadrupole moment functions taken from the literature. The infrared spectrum of the CO molecule in the heme pocket is calculated using the dipole moment time autocorrelation function and shows good agreement with experiment. In particular, the new model reproduces the experimentally observed splitting of the CO absorption spectrum. The splitting of 3-7 cm(-1) (compared to the experimental value of 10 cm(-1)) can be directly attributed to the two possible orientations of CO within the docking site at the edge of the distal heme pocket (the B states), as previously suggested on the basis of experimental femtosecond time-resolved infrared studies. Further information on the time evolution of the position and orientation of the CO molecule is obtained and analyzed. The calculated difference in the free energy between the two possible orientations (Fe...CO and Fe...OC) is 0.3 kcal mol(-1) and agrees well with the experimentally estimated value of 0.29 kcal mol(-1). A comparison of the new fluctuating charge model with an established fixed charge model reveals some differences that may be critical for the correct prediction of the infrared spectrum and energy barriers. The photodissociation of CO from the myoglobin mutant L29F using the new model shows rapid escape of CO from the distal heme pocket, in good agreement with recent experimental data. The effect of the protein environment on the multipole moments of the CO ligand is investigated and taken into account in a refined model. Molecular dynamics simulations with this refined model are in agreement with the calculations based on the gas-phase model. However, it is demonstrated that even small changes in the electrostatics of CO alter the details of the dynamics.

Project description:Infrared spectra of heme-bound CO in sperm whale carbonmonoxy myoglobin and two mutants (H64L and H97F) were studied in the pH range from 4.2 to 9.5. Comparison of the native protein with the mutants shows that the observed pH effects can be traced to protonations of two histidine residues, H64 and H97, near the active site. Their imidazole sidechains experience simple, uncoupled Henderson-Hasselbalch type protonations, giving rise to four different protonation states. Because two of the protonation states are linked by a pH-independent equilibrium, the overall pH dependence of the spectra is described by a linear combination of three independent components. Global analysis, based on singular value decomposition and matrix least-squares algorithms enabled us to extract the pK values of the two histidines and the three basis spectra of the protonating species. The basis spectra were decomposed into the taxonomic substates A(0), A(1), and A(3), previously introduced in a heuristic way to analyze CO stretch spectra in heme proteins at fixed pH (see for instance, Biophys. J. 71:1563-1573). Moreover, an additional, weakly populated substate, called A(x), was identified. Protonation of H97 gives rise to a blue shift of the individual infrared lines by about 2 cm(-1), so that the A substates actually appear in pairs, such as A(0) and A(0)(+). The blue shift can be explained by reduced backbonding from the heme iron to the CO. Protonation of the distal histidine, H64, leads to a change of the infrared absorption from the A(1) or A(3) substate lines to A(0). This behavior can be explained by a conformational change upon protonation that moves the imidazole sidechain of H64 away from the CO into the high-dielectric solvent environment, which avoids the energetically unfavorable situation of an uncompensated electric charge in the apolar, low-dielectric protein interior. Our results suggest that protonation reactions serve as an important mechanism to create taxonomic substates in proteins.

Dataset Information

Primary protein response after ligand photodissociation in carbonmonoxy myoglobin.

Publications

Primary protein response after ligand photodissociation in carbonmonoxy myoglobin.

Similar Datasets

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