Ultrafast Pump–Probe Study of the Excited-State Charge-Transfer Dynamics in Blue Copper Rusticyanin

Abstract

We have used femtosecond pump-probe spectroscopy to investigate the excited-state dynamics of the anticancer blue copper protein rusticyanin, by exciting its ligand to metal charge-transfer band with 25 fs pump pulses centered at 585 nm. The charge-transfer excited state decays exponentially to the ground state with a time constant of about 230 fs, and its recovery is modulated by coherent oscillations. The Fourier transform of the oscillatory component of the signal provides most of the vibrational modes obtained by means of conventional resonance Raman studies, in addition to the low frequency modes below 80 cm(-1) believed to reflect collective motions of biological relevance.

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

“…Since these electronic transitions involve the movement of charge from ligand atoms to the copper center, the decay of excited state population can be likely assigned to a return charge transfer process which is called metal-to-ligand chargetransfer (MLCT). The constant offset (corresponding to the almost infinite lifetime exponential decay) could be the evidence of an amount of population (around 20%) becoming trapped in a long-living state or on the excited-state surface, preventing the reestablishment of equilibrium on the time-scale of the pump-probe experiment, as suggested in similar studies in which a constant offset was required to describe the single wavelength time traces [11,[14][15][16][17][18]. These results allow us to say that after light induced LMCT excitation in POXC a single-step deactivation process with a time constant of 375 fs (in terms of a hole transfer representation [11]: S(Cys-π) → 375 fs Cu d x 2 −y 2 ) occurs; thus POXC features a deactivation process similar to that of azurin [14], some plastocyanins [15,18], umecyanin [16] and rustycianin [17].…”

“…The constant offset (corresponding to the almost infinite lifetime exponential decay) could be the evidence of an amount of population (around 20%) becoming trapped in a long-living state or on the excited-state surface, preventing the reestablishment of equilibrium on the time-scale of the pump-probe experiment, as suggested in similar studies in which a constant offset was required to describe the single wavelength time traces [11,[14][15][16][17][18]. These results allow us to say that after light induced LMCT excitation in POXC a single-step deactivation process with a time constant of 375 fs (in terms of a hole transfer representation [11]: S(Cys-π) → 375 fs Cu d x 2 −y 2 ) occurs; thus POXC features a deactivation process similar to that of azurin [14], some plastocyanins [15,18], umecyanin [16] and rustycianin [17]. In fact, by using the analysis of single time traces detected at specific wavelengths, a 270 fs decay time for the ground state recovery was observed in plastocyanin from Synechococcus PCC7942 when excited with a 33 fs pulse at 635 nm [18], and also in Pseudomonas aeruginosa azurin [14] and poplar plastocyanin [15] by exciting the sample with a 10 fs pulse centered at 550 nm (not exactly in resonance with the LMCT bands which instead show the maximum at around 630 and 600 nm for azurin and plastocyanin, respectively) and analyzing the differential transmission at 580 and 560 nm in the case of azurin and plastocyanin, respectively.…”

“…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]). 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].…”

“…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]).…”