Nuclear resonance vibrational spectroscopy (NRVS) is an emerging site-specific probe of active site vibrational dynamics in metalloproteins. 1,2 NRVS is a synchrotron-based technique that uniquely targets the vibrations of a Mössbauer nucleus, such as 57 Fe, without interference from vibrations of other atoms, and reveals not only the frequency, but also the (mean squared) amplitude, 1b,2a of all vibrations of the probe nucleus along the direction of the incident X-ray beam. Quantitative characterization of vibrational modes involving a reactive probe atom can illuminate mechanisms of complex biomolecules.Reactions with heme proteins mediate the physiological effects of nitric oxide (NO). The proposed trigger for activation of soluble guanylate cyclase (sGC) is rupture of the covalent Fe-His bond 3a,b in the heme-containing domain 3c,d upon NO binding to the Fe. A thermodynamic consequence of NO-induced weakening of a trans Fe-imidazole bond, as observed in several heme systems, is that imidazole binding should weaken a trans Fe-NO bond. Model compound structures support such a reciprocal negative trans interaction, 4a although protein structural data 4b-d may not have sufficient precision to resolve the 3 pm increase in Fe-NO bond length due to imidazole binding.On the other hand, vibrational frequencies respond sensitively to bond length changes of this magnitude, and it is therefore puzzling that the frequency attributed to stretching of the Fe-NO bond in six-coordinate imidazole-ligated heme proteins 5 is higher, rather than lower, than the frequencies observed for five-coordinate iron nitrosyl hemes. For example, the assigned Fe-NO stretching frequencies of the 5-and 6-coordinate NO complexes with myoglobin (MbNO) are 521 cm −1 and 552 cm −1 , respectively. 5c NRVS measurements on 6-coordinate MbNO suggest reexamination of this issue (Fig. 1A). The Fe-weighted vibrational density of states (VDOS) D(ν) samples the vibrational kinetic energy distribution (KED), with each mode contributing an area e Fe 2 equal to the fraction of mode energy associated with Fe motion. 2a,d The e Fe 2 = 0.11 area of the feature at 547 cm −1 is well below the e Fe 2 = 0.23-0.33 range that we observed for the FeNO stretching mode in a series of 5-coordinate nitrosyl porphyrins, 2d but is clearly visible because of the distinctly improved signal quality compared to previously published NRVS measurements on myoglobin. 1a,b,d In contrast, a mode with an area e Fe 2 = 0.25 appears at 443 cm −1 , near a
We use nuclear resonance vibrational spectroscopy and computational predictions based on density functional theory (DFT) to explore the vibrational dynamics of (57)Fe in porphyrins that mimic the active sites of histidine-ligated heme proteins complexed with carbon monoxide. Nuclear resonance vibrational spectroscopy yields the complete vibrational spectrum of a Mössbauer isotope, and provides a valuable probe that is not only selective for protein active sites but quantifies the mean-squared amplitude and direction of the motion of the probe nucleus, in addition to vibrational frequencies. Quantitative comparison of the experimental results with DFT calculations provides a detailed, rigorous test of the vibrational predictions, which in turn provide a reliable description of the observed vibrational features. In addition to the well-studied stretching vibration of the Fe-CO bond, vibrations involving the Fe-imidazole bond, and the Fe-N(pyr) bonds to the pyrrole nitrogens of the porphyrin contribute prominently to the observed experimental signal. All of these frequencies show structural sensitivity to the corresponding bond lengths, but previous studies have failed to identify the latter vibrations, presumably because the coupling to the electronic excitation is too small in resonance Raman measurements. We also observe the FeCO bending vibrations, which are not Raman active for these unhindered model compounds. The observed Fe amplitude is strongly inconsistent with three-body oscillator descriptions of the FeCO fragment, but agrees quantitatively with DFT predictions. Over the past decade, quantum chemical calculations have suggested revised estimates of the importance of steric distortion of the bound CO in preventing poisoning of heme proteins by carbon monoxide. Quantitative agreement with the predicted frequency, amplitude, and direction of Fe motion for the FeCO bending vibrations provides direct experimental support for the quantum chemical description of the energetics of the FeCO unit.
The biological importance of nitric oxide has changed from that of toxic gas to that of an essential cellular signaling agent. 1 In many of these processes, the binding of NO to a heme protein and the labilization of the ligand trans to NO or another rearrangement is the significant signaling event. Proposed mechanisms for the heme proteins cytochrome c′ and soluble guanylate cyclase have presented a scenario in which the coordination to heme changes during the physiological cycle. 2 Understanding how these coordination events impact already coordinated ligands (and vice versa) is an important step in understanding the heme-NO interaction. At the literal center of these studies is the heme iron, whose motion along the coordinate axes is associated with reactive modes such as the ν Fe -Im , ν Fe-NO /δ Fe-N-O and heme doming.In this communication, we examine whether ligand orientation and bond distance changes substantially modulate iron dynamics in six-coordinate [Fe(Porph)(1-MeIm)(NO)] 3 derivatives. This study has been facilitated by the isolation of two crystalline polymorphs of [Fe(TpFPP)(1-MeIm)(NO)] that display differing room temperature solid-state ν N-O (1631 cm -1 and 1640 cm -1 ). Additional vibrational data for the polymorphic forms is available from nuclear resonance vibrational measurements (NRVS). Briefly, NRVS is a novel technique that provides information on all vibrational frequencies for which there is iron motion. 4 Spectra were obtained on powder samples of the two species and are displayed in Figure S1. The powder data clearly shows differences in the overall iron vibrational modes. We have also obtained oriented single crystal NRVS data on both species that provides detailed information on the character of the modes, which allows for a detailed examination of the differences between the two. An analysis of their molecular structures and vibrational data yields a detailed view of how different molecular structure features affect the dynamics of the iron atom.The crystalline polymorphs are in the triclinic and monoclinic crystal systems; both are isolated from the same crystallization experiments. The two crystal types are illustrated in Figure S2. Figure 1 illustrates the two molecular structures. The FeNO and imidazole planes are within 1° of coplanarity (monoclinic form) and within 25° (triclinic form). The relative orientation of the imidazole N-CH 3 bond and the bent FeNO group are of the opposite sense in the two species; the monoclinic form has a ″cisoid″ arrangement and the triclinic a ″transoid″ one. The two NO ligands make angles of 38.5° and 43.2° with the closest Fe-N p vector, so that when the four porphyrin nitrogen atoms are superimposed, the NO ligands are almost superimposed (see Figure S3). Major differences include relative rotations of the two imidazole ligands, the relative sense of NO and imidazole directions, small differences in the trans Fe-N Im bond distance, core conformations and positions of the peripheral p-fluorophenyl groups. The changing peripheral group direct...
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