Femtosecond coherent transient infrared spectroscopy of reaction centers from Rhodobacter sphaeroides.

Abstract

Protein and cofactor vibrational dynamics assodated with photoexcitation and charge separation in the photosynthetic reaction center were investigated with femtosecond (300-400 fs) time-resolved inaed (1560-1960 cm-') spectroscopy. The experiments are in the coherent transient limit where the quantum uncertainty principle governs the evolution of the protein vibrational changes. No significant protein relaxation accompanies charge separation, although the electric field resulting from charge separation m e the polypeptide carbonyl spectra. The potential energy surfaces of the "spedal pair" P and the photoexcited singlet state P* and environmental perturbations on them are similar as judged from coherence transfer measurements. The vibrational dephasing time of P* modes in this region is 600 fs. A subpicosecond transient at 1665 cm-1 was found to have the kinetics expected for a sequential electron transfer process.Kinetic signatures of all other transient intermediates, P. P*9and P+, participating in the primary steps of photosynthesis were identified in the difference infrared spectra.The reaction center (RC) protein contains about a thousand residues in three different subunits and has a molecular weight of -100,000. Two of the subunits, L and M, are related by an approximate C2 symmetry (1-5). Each of these consists of seven transmembrane helices that span the -45-A membrane lipid bilayer. Together these two subunits hold four bacteriochlorophyll a (BChl) molecules, two bacteriopheophytins (Bphs), two quinones (Q), and one nonheme iron atom. The C2 axis passes through the iron atom (near the cytoplasmic side of the membrane) and through the midpoint of the line joining the centers of the two BChl molecules on the periplasmic side that are in van der Waals contact and constitute the "special pair" (P).The primary kinetic event of the photosynthetic process consists of the transfer of an electron down the transmembrane chain of cofactors against the membrane potential using the energy ofan absorbed photon (for a review, see ref. (23) have presented evidence for a two-step electron transfer scheme where P* reduces BChl in 2.8 ps and the BChl-species decays with a time constant of 0.8 ps to form P+Bph-. Theoretical calculations are not unequivocal as to the involvement of BCh1-(24). Finally, information on the participation of the protein medium in the electron transfer is scarce.Ultrafast vibrational spectroscopy is a natural approach to obtain answers to the foregoing questions. The protein IR absorbance before and after the electron transfer should manifest the changes that accompany the charge separation. Indeed studies of trapped intermediates (refs. 25-27 cm-'. The electron transfer was initiated with a pulse of 870-nm light with energy 0.5 PJ and probed with a tunable carbon monoxide laser, which was gated (29, 30) by a short 870-nm pulse. The femtosecond pulses are generated by a mode-locked titanium: A1203 oscillator (MIRA-900; Coherent Radiation, Palo Alto, CA). A regenerative amplifier wa...

Identification of the First Steps in Charge Separation in Bacterial Photosynthetic Reaction Centers of Rhodobacter sphaeroides by Ultrafast Mid-Infrared Spectroscopy: Electron Transfer and Protein Dynamics

“…Time-resolved mid-IR spectroscopy also opens up the unique possibility of investigating the protein response that follows electron transfer, and therefore the relaxation processes that occur in the vicinity of the cofactors on the timescale of electron transfer. For the bacterial RC, several studies of the initial electron transfer dynamics and protein response to this process have been reported (19)(20)(21)(22)(23)(24)(25). Hamm et al (19) carried out measurements on Rb.…”

Reorganization Energy of the Initial Electron-Transfer Step in Photosynthetic Bacterial Reaction Centers

“…The reaction generates a P ϩ H Ϫ ion pair with a time constant of ϳ3 ps at room temperature, and it becomes even faster at cryogenic temperatures (Woodbury et al, 1985;Breton et al, 1988;Fleming et al, 1988). Recent experimental work has provided increasing support for the view that charge separation occurs in two distinct steps (Holzapfel et al, 1990;Arlt et al, 1993Arlt et al, , 1996Shkuropatov and Shuvalov, 1996;Maiti et al, 1994;Schmidt et al, 1994Schmidt et al, , 1995Heller et al, 1995Heller et al, , 1996Kirmaier et al, 1995a,b;Holzwarth and Muller, 1996;Van Brederode et al, 1996;Kennis et al, 1997). In the first step, P* probably transfers an electron to a neighboring bacteriochlorophyll (B), forming a P ϩ B Ϫ ion pair; in the second step, an electron moves from B Ϫ to H:…”

Conformational Changes of Arginine Kinase Induced by Photochemical Release of Nucleotides from Caged Nucleotides

“…In this respect, site-directed mutagenesis, chemical modifications and comparisons with known three-dimensional structures (solved by X-ray diffraction) of parent enzymes, such as mitochondria1 creatine kinase (Fritz-Wolf et al, 1996), could be used to gain more details on the assignments of infrared bands, and on the molecular mechanism of phosphate transfer from ATP to Arg. Furthermore, the catalytic process could be investigated with time-resolved infrared spectroscopy (Hienerwadel et al, 1995;Maiti et al, 1994;Sasaki et al, 1995;Weidlich et al, 1994) or with modulation spectroscopy (Fringeli, …”