Ultrafast Pump−Probe Study of Excited-State Charge-Transfer Dynamics in Umecyanin from Horseradish Root

Abstract

We have applied femtosecond pump-probe spectroscopy to investigate the excited-state dynamics of umecyanin from horseradish roots, by exciting its 600-nm ligand-to-metal charge-transfer band with a 15-fs pulse and probing over a broad range in the visible region. The decay of the pump-induced ground-state bleaching is modulated by clearly visible oscillations and occurs exponentially with a time constant depending on the observed spectral component of the transmission difference signal, ranging from 270 fs up to 700 fs. The slower decaying process characterizes the spectral component corresponding to the metal-to-ligand charge-transfer transition. The excited-state decay rate is significantly lower than in other blue copper proteins, probably because of the larger energy gap between ligand- and metal-based orbitals in umecyanin. Wavelength dependence of the recovery times could be due to either the excitation of several transitions or the occurrence of intramolecular vibrational relaxation within the excited state. We also find evidence of a hot ground-state absorption, at 700 nm, persisting for several picoseconds. The vibrational coherence induced by the ultrashort pump pulse allows vibrational activity to be observed, mainly in the ground state, as expected in a system with fast excited-state decay. However, we find evidence of a rapidly damped oscillation, which we assign to the excited state. Finally, the Fourier transform of the oscillatory component of the signal presents additional bands in the low-frequency region which are assigned to collective motions of the protein.

Ultrafast excited-state charge-transfer dynamics in laccase type I copper site

“…This well correlates with previous experiments on T1 Cu site-containing proteins in which the ΔT / T time traces extracted from a 2D map were usually analyzed in terms of a sum of decaying exponentials plus a constant offset [16,20]. We can therefore conclude that, after the light-induced excitation of the LMCT T1 Cu site transition, a single deactivation process with a lifetime of 375 ± 25 fs occurs.…”

“…At variance, a single step deactivation process (with a time constant of 270 fs) has been observed for plastocyanin from Synechococcus PCC7942 [18], azurin [14], and poplar plastocyanin [15], while a slightly faster single step process has been detected for the rusticyanin CT transition (time constant of 230 fs) [17]. The dynamics of the CT excited state in umecyanin from Horseradish root has also been described as a single step ground state population recovery process, with probe wavelength-dependent time constants (from 470 to 700 fs) [16]. In all the cases, the knowledge of the excited state dynamics has helped in better describing the overall CT process and the copper site geometry.…”

“…The electrons are then shuttled through the highly conserved His-Cys-His tripeptide to the TNC, where O 2 is reduced to water [2,3]. Ultrafast resonant pump-probe spectroscopy is a powerful tool to investigate the charge transfer processes in copper proteins and the relationship between enzyme structure and functionalities [11][12][13][14][15][16][17][18][19]. In these experiments, the protein is excited in resonance with the absorption band by an ultrashort pump pulse that creates a population in the excited electronic state and vibrational coherences in both the ground and excited states.…”

“…In this way excited-state population dynamics and real-time analysis of vibrational motions coupled to electronic transitions are investigated. By resonant pump-probe spectroscopy the CT dynamics occurring in T1 Cu site of various cupredoxins has been investigated [11,[14][15][16][17][18][19]. The excited state dynamics of some plastocyanins has been described as a multi-step relaxation process (two-step process in spinach plastocyanin, with characteristic times of 125 and 285 fs for the first and second steps, respectively [11]; three-step deactivation for Silene pratensis plastocyanin, with time constants of 40, 90 and 250 fs [19]).…”

Optical investigation of the electron transfer protein azurin–gold nanoparticle system

“…Interestingly, it has been shown that an electron transfer well mimicking the thermal one can be induced by light irradiating Az in its LMCT band [25], similarly to other blue copper proteins [26][27][28], pointing out an intriguing interplay between Az optical and electrical properties. Additionally, Az ET path is hypothesized to involve the protein single triptophan residue [29], an intrinsic fluorophore located at position 48 (Trp48) in a highly hydrophobic environment [30,31], which is very sensitive to protein conformation [30][31][32].…”