Resonance Raman detection of structural dynamics at the active site in hemoglobin.

Abstract

The iron-histidine stretching mode in deoxyhemoglobin displays a large change in frequency and width upon lowering the temperature from 300 to 10 K. The temperature dependence ofthe data indicates the presence of dynamic processes. The dynamics of this mode in frozen hemoglobins can be qualitatively and quantitatively described as a vibrational dephasing via anharmonic coupling to other vibrations of the heme-imidazole system. The effects that occur at the melting transition in the low frequency modes cannot be quantitatively addressed at this point but may be indicative of the introduction of additional degrees of freedom predicated on protein influences that reflect differences in protein quaternary structure.Large amplitude motions of atoms and groups of atoms have recently been detected in proteins by crystallographic (1, 2) and spectroscopic (3-5) techniques. Although in some instances the functional importance of these motions has not yet been assessed, in others the importance is clear. For example, the heme binding sites in hemoglobin and myoglobin are only accessible because molecular motions occur that modulate the size of the narrow entry channel and thus allow the penetration of small ligands (6). These motions involve both transitions between local minima in the potential energy surface (conformational substates) and thermal population of excited levels. A picture emerges of fluid-like proteins in which molecular groups rotate about single bonds, hydrogen bonds break and reform, and new Van der Waals interactions occur continuously.There have been few reports in which dynamic processes in biomolecules have been probed by Raman scattering (7-10) although its potential for such studies is widely recognized because the Raman frequencies associated with bonds that connect molecular groupings are sensitive to structural dynamics. Resonance Raman scattering studies of dynamic processes in heme proteins are especially promising. The technique can be a sensititive probe of the properties of the active sites where the existence of structural dynamics could have direct functional importance.We report here a resonance Raman investigation of hemoglobin in which we have detected a temperature-dependent variation in the width and position of the mode assigned as the iron-histidine (Fe-His) stretching motion. We interpret these results as evidence for structural dynamics involving fluctuations in the Fe-His bond energy due to anharmonic coupling between modes.METHODS AND MATERIALS Raman scattering data from solutions above and just below freezing were obtained with previously described Raman difference instrumentation (11). Samples were placed in glass or fused silica rotating cells and the scattered light was gathered with 900 collecting optics. For the low temperature studies (< -500C), the rotating liquid cell was removed and liquid samples were placed on the cold finger of a Heli-Tran Cryostat (Air Products and Chemicals, Allentown, PA) where they were frozen either by direct insertion into liquid nit...

Time‐resolved resonance Raman studies of carp hemoglobin

“…In human deoxy-hemoglobins (normal, mutant and chemically modified) the Fe-His stretching mode shifts from -215 cm -l for the T-state species to -221 +_ 3 cm -I for the R-state species [4,5]. In this study as well as in [11] similar frequencies were observed for the low-and highaffinity forms of deoxy-Hb (carp).…”

“…By focusing on the frequency of the iron-proximal histidine (F8) (Fe-His) stretching mode it is seen [2,3] that ligand binding causes a change in the heme-proximal histidine linkage that results in an increased frequency for the Fe-His stretching mode in the photolyzed species (at 10 ns) relative to that of the corresponding deoxy species. For both the deoxy [4,5] and the photolyzed [2,3] species the R state Hb's have the higher frequency. Here, we show that this same type of structural response, of the binding site to the ligand as well as the sensitivity of this response to quaternary structure is also in evidence in carp hemoglobin.…”

Characterization of the Proximal Ligand in the P420 Form of Inducible Nitric Oxide Synthase

“…As shown in Figure 6, for Mb, a down-shift in the ν Fe-His frequency was observed in D 2 O. The H 2 O-D 2 O isotopic shift was calculated to be 1.5 cm −1 based on the method reported previously,58 with the assumption that the ν Fe-His mode can be described by a Gaussian function 59. The same experiment was performed with iNOS P420 , but no isotopic shift was detected for the analogous 221 cm −1 mode.…”

Transient Raman Study of Hemoglobin: Structural Dependence of the Iron-Histidine Linkage

“…6 (1982)]. 8. In several pilot studies, we had used limited exposure (100 to 200 msec) compared with Ito 2-second exposures.…”