Dan Ionascu

Temperature-dependent heme kinetics with nonexponential binding and barrier relaxation in the absence of protein conformational substates

We present temperature-dependent kinetic measurements of ultrafast diatomic ligand binding to the “bare” protoheme (L1-FePPIX-L2, where L1 = H2O or 2-methyl imidazole and L2 = CO or NO). We found that the binding of CO is temperature-dependent and nonexponential over many decades in time, whereas the binding of NO is exponential and temperature-independent. The nonexponential nature of CO binding to protoheme, as well as its relaxation above the solvent glass transition, mimics the kinetics of CO binding to myoglobin (Mb) but on faster time scales. This demonstrates that the nonexponential kinetic response observed for Mb is not necessarily due to the presence of protein conformational substates but rather is an inherent property of the solvated heme. The nonexponential kinetic data were analyzed by using a linear coupling model with a distribution of enthalpic barriers that fluctuate on slower time scales than the heme–CO recombination time. Below the solvent glass transition (Tg ≈ 180 K), the average enthalpic rebinding barrier for H2O-PPIX-CO was found to be ≈1 kJ/mol. Above Tg, the barrier relaxes and is ≈6 kJ/mol at 290 K. Values for the first two moments of the heme doming coordinate distribution extracted from the kinetic data suggest significant anharmonicity above Tg. In contrast to Mb, the protoheme shows no indication of the presence of “distal” enthalpic barriers. Moreover, the wide range of Arrhenius prefactors (109 to 1011 s−1) observed for CO binding to heme under differing conditions suggests that entropic barriers may be an important source of control in this class of biochemical reactions.

Two-color pump-probe laser spectroscopy instrument with picosecond time-resolved electronic delay and extended scan range

An electronically delayed two-color pump-probe instrument was developed using two synchronized laser systems. The instrument has picosecond time resolution and can perform scans over hundreds of nanoseconds without the beam divergence and walk-off effects that occur using standard spatial delay systems. A unique picosecond Ti:sapphire regenerative amplifier was also constructed without the need for pulse stretching and compressing optics. The picosecond regenerative amplifier has a broad wavelength tuning range, which suggests that it will make a significant contribution to two-color pump-probe experiments. To test this instrument we studied the rotational correlation relaxation of myoglobin (τr=8.2±0.5ns) in water as well as the geminate rebinding kinetics of oxygen to myoglobin (kg1=1.7×1011s−1, kg2=3.4×107s−1). The results are consistent with, and improve upon, previous studies.

Coherence Spectroscopy Investigations of the Low-Frequency Vibrations of Heme: Effects of Protein-Specific Perturbations

Femtosecond coherence spectroscopy is used to probe the low-frequency (20-200 cm(-1)) vibrational modes of heme proteins in solution. Horseradish peroxidase (HRP), myoglobin (Mb), and Campylobacter jejuni globin (Cgb) are compared and significant differences in the coherence spectra are revealed. It is concluded that hydrogen bonding and ligand charge do not strongly affect the low-frequency coherence spectra and that protein-specific deformations of the heme group lower its symmetry and control the relative spectral intensities. Such deformations potentially provide a means for proteins to tune heme reaction coordinates, so that they can perform a broad array of specific functions. Native HRP displays complex spectral behavior above approximately 50 cm(-1) and very weak activity below approximately 50 cm(-1). Binding of the substrate analog, benzhydroxamic acid, leads to distinct changes in the coherence and Raman spectra of HRP that are consistent with the stabilization of a heme water ligand. The CN derivatives of the three proteins are studied to make comparisons under conditions of uniform heme coordination and spin-state. MbCN is dominated by a doming mode near 40 cm(-1), while HRPCN displays a strong oscillation at higher frequency (96 cm(-1)) that can be correlated with the saddling distortion observed in the X-ray structure. In contrast, CgbCN displays low-frequency coherence spectra that contain strong modes near 30 and 80 cm(-1), probably associated with a combination of heme doming and ruffling. HRPNO displays a strong doming mode near 40 cm(-1) that is activated by photolysis. The damping of the coherent motions is significantly reduced when the heme is shielded from solvent fluctuations by the protein material and reduced still further when T approximately < 50 K, as pure dephasing processes due to the protein-solvent phonon bath are frozen out.

Wavelength selective modulation in femtosecond pump-probe spectroscopy and its application to heme proteins

We demonstrate novel lock-in detection techniques, using wavelength selective modulation of ultrafast pump and probe laser pulses, to discriminate between vibrational coherence and electronic population decay signals. The technique is particularly useful in extracting low frequency oscillations from the monotonically decaying background, which often dominates the signal in resonant samples. The central idea behind the technique involves modulating the red and/or blue wings of the laser light spectrum at different frequencies, ΩR and ΩB, followed by a lock-in detection at the sum or difference frequency, ΩR±ΩB. The wavelength selective modulation and detection discriminates against contributions to the pump–probe signal that arise from degenerate electric field interventions (i.e., only field interactions involving different optical frequencies are detected). This technique can be applied to either the pump or the probe pulse to enhance the off-diagonal terms of the pump induced density matrix, or to selec...

Rapid timescale processes and the role of electronic surface coupling in the photolysis of diatomic ligands from heme proteins

We have observed coherent oscillations of the heme protein myoglobin (Mb) following femtosecond laser excitation and photodissociation of the CO, O2, and NO bound ligands. Use of a novel methodology, involving wavelength selective modulation of the pump and/or probe laser pulse train, allows us to discriminate between coherences created by pump fields of differing wavelength within the laser pulse versus signals that arise from the decay of either vibrational or electronic populations. The population driven signals appear when pump field interactions having the same optical frequency are allowed to contribute to the signal detection channel. One surprising result, which will be stressed in the discussion, is the observation of a distinct product state vibrational coherence (the iron-histidine stretching vibration of deoxy Mb at 220 cm(-1)) that depends upon the presence of pump field interactions having a wavelength mismatch that is equal to the 220 cm(-1) vibrational frequency. This observation is surprising because the iron-histidine mode is not observed in the resonance Raman measurements on the six-coordinate reactant species. Thus, the pump-pulse laser excitation between the ground and excited state, which leads to the ligand dissociation, is evidently able to create a field driven vibrational coherence of a resonance Raman inactive mode that extends into non-vertical regions of the reactive excited state potential energy surface. Non-radiative electronic surface crossing, followed by the rapid development of new electronic forces on the nuclei, appears to be ruled out as a source of the coherent signals (the random phase of the optically uncoupled modes is one possible explanation for this observation). The extremely rapid timescale (<< 150 fs) for the development of the (S = 2) high-spin product state of the iron atom from the initial unphotolyzed state (S = 0) is worthy of further theoretical discussion because of the spin forbidden nature of such a transition. Excited state admixtures of the iron spin states are presumably involved, and the mixing of these states, along with the unpaired electron on NO, may help to explain the ultrafast time scales and large amplitudes that characterize the NO geminate recombination in comparison to CO.

Low-frequency dynamics of Caldariomyces fumago chloroperoxidase probed by femtosecond coherence spectroscopy

Ultrafast laser spectroscopy techniques are used to measure the low-frequency vibrational coherence spectra and nitric oxide rebinding kinetics of Caldariomyces fumago chloroperoxidase (CPO). Comparisons of the CPO coherence spectra with those of other heme species are made to gauge the protein-specific nature of the low-frequency spectra. The coherence spectrum of native CPO is dominated by a mode that appears near 32-33 cm(-1) at all excitation wavelengths, with a phase that is consistent with a ground-state Raman-excited vibrational wavepacket. On the basis of a normal coordinate structural decomposition (NSD) analysis, we assign this feature to the thiolate-bound heme doming mode. Spectral resolution of the probe pulse (detuned detection) reveals a mode at 349 cm(-1), which has been previously assigned using Raman spectroscopy to the Fe-S stretching mode of native CPO. The ferrous species displays a larger degree of spectral inhomogeneity than the ferric species, as reflected by multiple shoulders in the optical absorption spectra. The inhomogeneities are revealed by changes in the coherence spectra at different excitation wavelengths. The appearance of a mode close to 220 cm(-1) in the coherence spectrum of reduced CPO excited at 440 nm suggests that a subpopulation of five coordinated histidine-ligated hemes is present in the ferrous state at a physiologically relevant pH. A significant increase in the amplitude of the coherence signal is observed for the resonance with the 440 nm subpopulation. Kinetics measurements reveal that nitric oxide binding to ferric and ferrous CPO can be described as a single-exponential process, with rebinding time constants of 29.4 +/- 1 and 9.3 +/- 1 ps, respectively. This is very similar to results previously reported for nitric oxide binding to horseradish peroxidase.

Femtosecond coherence spectroscopy using spectrally selective differential photodetection

Abstract We present a novel, spectrally selective, detection scheme for pump–probe femtosecond coherence spectroscopy (FCS) that attenuates the non-oscillatory background signal so that the coherent oscillations are more clearly revealed. The method is based on spectral dispersion of the probe beam after it has traversed the sample, followed by detection of spectrally specific portions with a differential detector containing two balanced photodiodes. By appropriately choosing the spectral windows detected by each photodiode, we show how vibrational oscillations can be selectively enhanced or suppressed. Information about the phase behavior of the vibrational modes is also revealed using this detection scheme.

Optical scanning instrument for ultrafast pump-probe spectroscopy of biomolecules at cryogenic temperatures

We demonstrate novel optical scanning and detection instrumentation that is particularly useful for the interrogation of stationary cryogenic samples in pump-probe spectroscopy. The technique uses a spinning lens to scan multiple laser beams over a stationary sample while maintaining the focal properties of the beams. This significantly lengthens the time window for the sample reset to equilibrium and improves the photostability of stationary samples. In addition, we describe a signal processing methodology that discriminates against the strong background signal that can arise from leakage of the pump laser pulse train into the detector. These techniques are particularly useful in pump-probe studies of ultrafast processes in biological systems where sample deterioration, pump induced thermal lensing, and light scattering into the detection channel (e.g., induced by light scattering from a cryogenic matrix) are problematic. Generally, the optical scanning and detection instrumentation described here enable...