Vibrational dynamics of biological molecules: Multi-quantum contributions

https://doi.org/10.1016/j.jpcs.2005.09.075Get rights and content

Abstract

High-resolution X-ray measurements near a nuclear resonance reveal the complete vibrational spectrum of the probe nucleus. Because of this, nuclear resonance vibrational spectroscopy (NRVS) is a uniquely quantitative probe of the vibrational dynamics of reactive iron sites in proteins and other complex molecules. Our measurements of vibrational fundamentals have revealed both frequencies and amplitudes of 57Fe vibrations in proteins and model compounds. Information on the direction of Fe motion has also been obtained from measurements on oriented single crystals, and provides an essential test of normal mode predictions. Here, we report the observation of weaker two-quantum vibrational excitations (overtones and combinations) for compounds that mimic the active site of heme proteins. The predicted intensities depend strongly on the direction of Fe motion. We compare the observed features with predictions based on the observed fundamentals, using information on the direction of Fe motion obtained either from DFT predictions or from single crystal measurements. Two-quantum excitations may become a useful tool to identify the directions of the Fe oscillations when single crystals are not available.

Introduction

Many essential biological processes take place at localized sites in proteins, which often incorporate metal ions. Site-selective vibrational spectroscopies [1], [2], [3], [4] have proven to be particularly revealing probes of the structure, dynamics and reactivity of protein active sites. Nuclear resonance vibrational spectroscopy (NRVS) [5], [6], [7], [8], [9], [10], [11], [12], [13], [14], [15], [16], [17], [18], [19], [20], [21], [22], [23], [24], [25], [26], [27], [28], [29] is a relatively new technique that takes advantage of the high brilliance and time-pulsed structure of third-generation synchrotron sources to reveal the complete vibrational spectrum of a probe nucleus. NRVS achieves the ultimate goal in selectivity, by targeting a single atom in a complex molecule. This method is particularly promising for characterizing the vibrational dynamics of 57Fe in biological macromolecules [10], [14], [20], [21], [24], [25], [26], and in small molecules designed to model protein active sites [15], [18], [19], [23], [27].

Unlike traditional vibrational spectroscopies, NRVS is highly quantitative, revealing not only the frequencies, but also the amplitudes of 57Fe vibrations. Measurements on oriented samples, such as single crystals, also yield the direction of vibrational motions [9], [11], [13], [17], [19], [22], [25], [27]. Other names used for this technique include phonon assisted Mössbauer effect [20], [21], [24], [25], inelastic X-ray scattering of synchrotron radiation [10], nuclear inelastic scattering [17], nuclear inelastic absorption [8], and nuclear resonant inelastic X-ray scattering [28].

One distinguishing feature of NRVS is the presence of multiquantum vibrational excitations, even for a strictly harmonic system. These multiquantum contributions are often removed numerically to determine a vibrational density of states [6], [16]. Chumakov and co-workers recently used multiquantum features to distinguish vibrational contributions from individual sites [29]. Here, we investigate two compounds, Fe(TPP)(2-MeIm) and Fe(TPP)(1-MeIm)(CO) (Fig. 1), designed to model the active sites of heme proteins. The high 57Fe concentrations achieved for these small molecules, relative to proteins, enable us to detect weak vibrational overtones and combinations. We explore the sensitivity of these two-quantum excitations to the direction of vibrational motion.

Section snippets

NRVS measurements

57Fe NRVS measurements were performed at sector 3-ID-D of the Advanced Photon Source at Argonne National Laboratory. A tunable high-resolution monochromator [31] selected X-rays in a 7 cm−1 (0.85 meV) energy interval near 14.4 keV, and an avalanche photodiode detected photons emitted from a sample placed in the resulting monochromatic beam. Rejection of events detected during a time window containing the arrival time of the X-ray pulse discriminated photons emitted over the 141 ns lifetime of the 57

One-quantum transitions

The top half of Fig. 2 displays the NRVS signal recorded for a randomly oriented polycrystalline sample of Fe(TPP)(1-MeIm)(CO) at 20 K. This compound is a simple model for the active site of heme proteins that bind the diatomic ligand CO. Previously, assignments for the vibrational fundamentals were proposed by adjusting the parameters in an empirical potential to reproduce the observed frequencies and amplitudes [23]. Two-quantum excitations are likely to contribute to a number of weak features

Conclusions

NRVS provides a detailed picture of the vibrational dynamics of Fe in biological molecules, including porphyrins and heme proteins. Measurements on oriented crystals and comparison with quantitative theoretical predictions enrich this picture with information on the direction of Fe motion and on the motion of other atoms, respectively. In addition to vibrational fundamentals, vibrational overtones and combinations contribute to the NRVS signal. The intensity of these two-quantum excitations

Acknowledgements

We are grateful for the loan of an Oxford cryocooler from BioCARS (sector 14 at the Advanced Photon Source) and for operating assistance provided by Dr T.-Y. Teng. We acknowledge financial support from the National Science Foundation (0240955 to J.T.S. and 9988763 to S.M.D.) and the National Institutes of Health (GM-52002 to J.T.S. and GM-38401 to W.R.S.). Use of the Advanced Photon Source was supported by the US Department of Energy, Basic Energy Sciences, Office of Science, under Contract No.

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      Citation Excerpt :

      NRVS yields quantitative values for vibrational amplitudes and directions, as well as frequencies, and thus provides a rigorous test of vibrational predictions. This information is proving particularly valuable for evaluating quantum chemical calculations based on density functional theory (DFT), which provide detailed predictions of many molecular ground state properties, including the optimized structure and a comprehensive description of vibrational dynamics (38,39,60,90,93–98). Here, we use NRVS measurements and DFT calculations to characterize the vibrational dynamics of ferrous carbonyl porphyrins, including [Fe(TPP)(1-MeIm)(CO)] (Fig. 1), that mimic the active site of six-coordinate CO-ligated heme proteins.

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