Ultrafast transient absorption studies on photosystem I reaction centers from Chlamydomonas reinhardtii. 2. Mutations near the P700 reaction center chlorophylls provide new insight into the nature of the primary electron donor

¹ Max-Planck-Inst. für Bioanorganische Chemie
² Max-Planck-Institut für Bioanorganische Chemie
³ Max-Planck-Institut für Strahlenchemie

^* To whom correspondence should be addressed. E-mail: holzwarth{at}mpi-muelheim.mpg.de.

Submitted on January 19, 2005
Revised on February 12, 2005
Accepted on 3 October 2005

	Abstract

The energy transfer and charge separation kinetics in several core photosystem I particles of Chlamydomonas reinhardtii with point mutations around the PA and PB reaction center chlorophylls (Chls) have been studied using ultrafast transient absorption spectroscopy in the femtosecond to nanosecond time range in order to characterize the influence on the early electron transfer processes. The data have been analyzed in terms of kinetic compartment models. The adequate description of the transient absorption kinetics requires three different radical pairs in the time range up to »100 ps. Also a charge recombination process from the first radical pair back to the excited state is present in all the mutants, as already shown previously for the wild type (Müller, M.G., J. Niklas, W. Lubitz, A. R. Holzwarth. Biophys.J. 85:3899-3922, 2003; and Holzwarth, A.R., Müller, M.G., Niklas J., Lubitz W., J.Phys. Chem. B, 109:5903-5911, 2005). In all mutants the primary charge separation occurs with the same effective rate constant within the error limits as in the wild type (»350 ns-1), which implies an intrinsic rate constant of charge separation of < 1 ps-1. The rate constant of the secondary electron transfer process is slowed down by a factor of approx. 2 in the mutant B-H656C which lacks the ligand to the central metal of Chl PB. For the mutant A-T739V, which breaks the hydrogen bond to the keto carbonyl of Chl PA, only a slight slowing down of the secondary electron transfer is observed. Finally for mutant A-W679A, which has the Trp near the PA Chl replaced, either no pronounced effect or, at best, a slight increase on the secondary electron transfer rate constants is observed. The effective charge recombination rate constant is modified in all mutants to some extent, with the strongest effect observed in mutant B-H656C. Our data strongly suggest that the Chls of the PA and PB pair, constituting what is traditionally called the "primary electron donor P700", are not oxidized in the first electron transfer process, but rather only in the secondary electron transfer step. We thus propose a new electron transfer mechanism for photosystem I where the accessory Chl(s) function as the primary electron donor(s) and the A0 Chl is the primary electron acceptor(s). This new mechanism also resolves in a straightforward manner the difficulty with the previous mechanism, where an electron would have to overcome a distance of approx. 14 Å in less than one ps in a single step. If interpreted within a scheme of single-sided electron transfer our data suggest that the B-branch is the active branch, although parallel A-branch activity can not be excluded. All the mutations do affect to a varying extent the energy difference between the reaction center excited state RC* and the first radical pair and thus affect the rate constant of charge recombination. It is interesting to note that the new mechanism proposed is in fact analogous to the electron transfer mechanism in photosystem II, where also the accessory Chl plays the role of the primary electron donor, rather than the "special Chl pair" P680 (Prokhorenko V. and A. R. Holzwarth. J.Phys.Chem.B 104:11563-11578, 2000)

Key Words: electron transfer, mutant, photosynthesis, photosystem I, reaction center, ultrafast spectroscopy

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