Observation of terahertz vibrations in Pyrococcus furiosus rubredoxin via impulsive coherent vibrational spectroscopy and nuclear resonance vibrational spectroscopy – interpretation by molecular mechanics

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Abstract

We have used impulsive coherent vibrational spectroscopy (ICVS) to study the Fe(S-Cys)4 site in oxidized rubredoxin (Rd) from Pyrococcus furiosus (Pf). In this experiment, a 15 fs visible laser pulse is used to coherently pump the sample to an excited electronic state, and a second <10 fs pulse is used to probe the change in transmission as a function of the time delay. PfRd was observed to relax to the ground state by a single exponential decay with time constants of ∼255–275 fs. Superimposed on this relaxation are oscillations caused by coherent excitation of vibrational modes in both excited and ground electronic states. Fourier transformation reveals the frequencies of these modes.

The strongest ICV mode with 570 nm excitation is the symmetric Fe–S stretching mode near 310 cm−1, compared to 313 cm−1 in the low temperature resonance Raman. If the rubredoxin is pumped at 520 nm, a set of strong bands occurs between 20 and 110 cm−1. Finally, there is a mode at ∼500 cm−1 which is similar to features near 508 cm−1 in blue Cu proteins that have been attributed to excited state vibrations.

Normal mode analysis using 488 protein atoms and 558 waters gave calculated spectra that are in good agreement with previous nuclear resonance vibrational spectra (NRVS) results. The lowest frequency normal modes are identified as collective motions of the entire protein or large segments of polypeptide. Motion in these modes may affect the polar environment of the redox site and thus tune the electron transfer functions in rubredoxins.

Introduction

Rubredoxins, with a single Fe(S-Cys)4 redox center, are among the smallest (∼50 amino acid) and simplest electron transfer proteins [1]. They are key electron donors in alkane oxidation pathways [2] and in superoxide reduction [3]. Apart from their biological significance, rubredoxins have often been employed as model systems for the development of spectroscopic techniques such as resonance Raman spectroscopy [4], [5], EXAFS [6], L-edge spectroscopy [7], X-ray magnetic circular dichroism – XMCD [8], high-resolution X-ray fluorescence [9], resonance energy transfer – RET [10], optically detected electron paramagnetic resonance – ODEPR [11], and nuclear resonance vibrational spectroscopy – NRVS [12], that are ultimately employed on more complex metalloproteins. Rubredoxin from Pyrococcus furiosus (PfRd), the object of the current study, has an unfolding rate of ∼10−6 s−1 at 100 °C [13] and has served as a model for structural features underlying thermal stability in hyperthermophilic proteins. An assortment of experimental [13], [14] and computational [15] investigations has examined PfRd large-scale dynamical properties with respect to both high temperature stability and the unusual temperature dependence of its redox potential. The redox potential is about 0 mV at 25 °C and changes to ∼160 mV at 90 °C at pH 8.0. Despite this wealth of studies, questions remain about its conformational flexibility, especially in regard to motion that could affect its electron transfer properties.

The structure of PfRd has been characterized by X-ray diffraction at 0.95 Å resolution (1BRF) [16], by neutron diffraction at 1.5 Å (1VCX) [17], and in solution by NMR [18]. The approximately tetrahedral FeS4 site has been described as approaching D2d symmetry via compression along an S4 axis (Chart 1). This distortion is exhibited in part as two shorter Fe–S bond lengths (2.25–2.26 Å) (Fe–SCys8 and Fe–SCys41) and two slightly longer Fe–S bonds (2.28–2.30 Å) (Fe–SCys5 and Fe–SCys38).

In this paper, we study the vibrational dynamics of oxidized PfRd using impulsive coherent vibrational spectroscopy (ICVS). This is a technique enabling direct time-domain observation of the vibrational modes coupled to an electronic transition. It involves pumping an optical transition in the sample with a short (∼10 fs) pulse of sufficient intensity to drive a fraction of the molecules of interest into the excited state, and then monitoring the pump-induced transmission changes with a suitably delayed short probe pulse. If the pump pulse has a duration significantly shorter than the periods of the vibrations of interest, it projects the ground-state multidimensional vibrational wave function onto the excited state in the form of a vibrational wave packet [19], [20]. Periodic motion of the localized packet formed in this way along displaced bond coordinates modulates in time the molecular absorption, which is measured by the probe pulse. Details of the ICVS theory have been explained elsewhere [21], [22].

ICVS has particular strengths with respect to other vibrational techniques – it enables direct time-domain observation of low frequency vibrational modes that are difficult to observe by far-infrared or Raman spectroscopies, it provides information on excited state vibrational modes which are not accessed by resonance Raman, and it is relatively insensitive to interference from fluorescent contaminants or solvent bands. ICVS has already been applied to heme proteins [23], [24], green fluorescent protein [25], and blue copper proteins such as azurin [26] and plastocyanin [27].

In this paper, we report on the real-time vibrational dynamics of oxidized PfRd pumped at 520 or 570 nm and probed at several different wavelengths. The ICV spectra extend to lower frequencies than typical metalloprotein resonance Raman spectra, revealing modes never seen before in Fe–S proteins, but similar to those seen in blue Cu proteins [27], [28] and in heme proteins by inelastic neutron scattering [29]. Normal mode calculations that include the entire protein as well as solvent water are used to interpret the experimental spectra. These calculations can be used to explain both the current ICVS observations and to quantitatively reproduce the recently reported NRVS data [12]. The results are also compared with previous Raman and molecular mechanics analyses of rubredoxins.

Section snippets

Protein purification and sample preparation

PfRd was purified by published procedures and concentrated to 12 mg/ml for shipping [30]. The protein was diluted to give an absorbance of 0.31 at 490 nm in a Tris buffer at pH 8. The ICVS measurements used 0.5 mm pathlength quartz cuvettes.

Impulsive coherent vibrational spectroscopy

ICVS time traces were recorded at room temperature using published procedures [27] at the National Laboratory for Ultrafast and Ultraintense Optical Science (Milano, Italy). The experimental setup starts with an amplified Ti:sapphire laser (500 μJ, 150 fs, 1 kHz)

Results

PfRd solutions were investigated by pump-probe experiments resonant with the S  Fe ligand-to-metal charge-transfer (LMCT) transitions, using 15 fs pump pulses centred at either 520 or 570 nm. For both pump wavelengths, different probe wavelengths (from 480 to 700 nm) of the ultra-broadband probe pulse were analyzed. Fig. 1 shows the ground-state absorption spectrum of PfRd together with the spectra of pump and probe pulses.

The time evolutions of the differential transmission (ΔT/T) signals,

Discussion and conclusions

The experimental results from our study of PfRd reveal a variety of previously unobserved low frequency vibrational modes, below 100 cm−1, that are observed in both the NRVS and ICVS data. The interpretation of these features has been assisted by a full protein normal mode analysis. Overall, these low frequency modes can be assigned to collective motion of the entire protein or to delocalized collective motion of large segments of polypeptide. These motions involve relative movement of the Fe–S

Abbreviations

    ICVS

    impulsive coherent vibrational spectroscopy

    Rd

    rubredoxin

    Pf

    Pyrococcus furiosus

    NRVS

    nuclear resonance vibrational spectroscopy

    EXAFS

    extended X-ray absorption fine structure

    XMCD

    X-ray magnetic circular dichroism

    RET

    resonance energy transfer

    NMR

    nuclear magnetic resonance

    NOPA

    non-collinear optical parametric amplifier

    ABNR

    adopted basis Newton–Raphson

    LMCT

    ligand-to-metal charge-transfer transition

    PB

    photobleaching

    PA

    photoinduced absorption

    SWFT

    sliding window Fourier transform

    PVDOS

    partial vibrational density

Acknowledgments

This work was funded by NIH Grants EB-001962 (SPC), GM-65440 (SPC), GM-60329 (MWWA), GM-45303 (TI), and the DOE Office of Biological and Environmental Research (SPC). This work has been partially supported by the FIRB-MIUR Project “Molecular Nanodevices” and a PRIN-MIUR 2004 project.

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