Xiong Ye

Ultrafast dynamics of diatomic ligand binding to nitrophorin 4

Nitrophorin 4 (NP4) is a heme protein that stores and delivers nitric oxide (NO) through pH-sensitive conformational change. This protein uses the ferric state of a highly ruffled heme to bind NO tightly at low pH and release it at high pH. In this work, the rebinding kinetics of NO and CO to NP4 are investigated as a function of iron oxidation state and the acidity of the environment. The geminate recombination process of NO to ferrous NP4 at both pH 5 and pH 7 is dominated by a single approximately 7 ps kinetic phase that we attribute to the rebinding of NO directly from the distal pocket. The lack of pH dependence explains in part why NP4 cannot use the ferrous state to fulfill its function. The kinetic response of ferric NP4NO shows two distinct phases. The relative geminate amplitude of the slower phase increases dramatically as the pH is raised from 5 to 8. We assign the fast phase of NO rebinding to a conformation of the ferric protein with a closed hydrophobic pocket. The slow phase is assigned to the protein in an open conformation with a more hydrophilic heme pocket environment. Analysis of the ultrafast kinetics finds the equilibrium off-rate of NO to be proportional to the open state population as well as the pH-dependent amplitude of escape from the open pocket. When both factors are considered, the off-rate increases by more than an order of magnitude as the pH changes from 5 to 8. The recombination of CO to ferrous NP4 is observed to have a large nonexponential geminate amplitude with rebinding time scales of approximately 10(-11)-10(-9) s at pH 5 and approximately 10(-10)-10(-8) s at pH 7. The nonexponential CO rebinding kinetics at both pH 5 and pH 7 are accounted for using a simple model that has proven effective for understanding CO binding in a variety of other heme systems (Ye, X.; et al. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 14682).

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.