The temperature dependence of the biphasic electron transfer (ET) from the secondary acceptor A1 (phylloquinone) to iron-sulfur cluster F(X) was investigated by flash absorption spectroscopy in photosystem I (PS I) isolated from Synechocystis sp. PCC 6803. While the slower phase (tau=340 ns at 295 K) slowed upon cooling according to an activation energy of 110 meV, the time constant of the faster phase (tau=11 ns at 295 K) was virtually independent of temperature. Following a suggestion in the literature that the two phases arise from bidirectional ET involving two symmetrically arranged phylloquinones, Q(K)-A and Q(K)-B, it is concluded that energetic parameters (most likely the driving forces) rather than the electronic couplings are different for ET from Q(K)-A to F(X) and from Q(K)-B to F(X). Two alternative schemes of ET in PS I are presented and discussed.

The core of photosystem I (PS1) is composed of the two related integral membrane polypeptides, PsaA and PsaB, which bind two symmetrical branches of cofactors, each consisting of two chlorophylls and a phylloquinone, that potentially link the primary electron donor and the tertiary acceptor. In an effort to identify amino acid residues near the phylloquinone binding sites, all tryptophans and histidines that are conserved between PsaA and PsaB in the region of the 10th and 11th transmembrane ␣-helices were mutated in Chlamydomonas reinhardtii. The mutant PS1 reaction centers appear to assemble normally and possess photochemical activity. An electron paramagnetic resonance (EPR) signal attributed to the phylloquinone anion radical (A 1 ؊ ) can be observed either transiently or after illumination of reaction centers with pre-reduced iron-sulfur clusters. Mutation of PsaA-Trp 693 to Phe resulted in an inability to photo-accumulate A 1 ؊ , whereas mutation of the analogous tryptophan in PsaB (PsaB-Trp 673 ) did not produce this effect. The PsaA-W693F mutation also produced spectral changes in the time-resolved EPR spectrum of the P 700 ؉ A 1 ؊ radical pair, whereas the analogous mutation in PsaB had no observable effect. These observations indicate that the A 1 ؊ phylloquinone radical observed by EPR occupies the phylloquinone-binding site containing PsaA-Trp 693 . However, mutation of either tryptophan accelerated charge recombination from the terminal Fe-S clusters.

Type I homodimeric reaction centers, particularly the class present in heliobacteria, are not well understood. Even though the primary amino acid sequence of PshA in Heliobacillus mobilis has been shown to contain an F(X) binding site, a functional Fe-S cluster has not been detected by EPR spectroscopy. Recently, we reported that PshB, which contains F(A)- and F(B)-like Fe-S clusters, could be removed from the Heliobacterium modesticaldum reaction center (HbRC), resulting in 15 ms lifetime charge recombination between P798(+) and an unidentified electron acceptor [Heinnickel, M., Shen, G., Agalarov, R., and Golbeck, J. H. (2005) Biochemistry 44, 9950-9960]. We report here that when a HbRC core is incubated with sodium dithionite in the presence of light, the 15 ms charge recombination is replaced with a kinetic transient in the sub-microsecond time domain, consistent with the reduction of this electron acceptor. Concomitantly, a broad and intense EPR signal arises around g = 5 along with a minor set of resonances around g = 2 similar to the spectrum of the [4Fe-4S](+) cluster in the Fe protein of Azotobacter vinelandii nitrogenase, which exists in two conformations having S = (3)/(2) and S = (1)/(2) ground spin states. The Mössbauer spectrum in the as-isolated HbRC core shows that all of the Fe is present in the form of a [4Fe-4S](2+) cluster. After reduction with sodium dithionite in the presence of light, approximately 65% of the Fe appears in the form of a [4Fe-4S](+) cluster; the remainder is in the [4Fe-4S](2+) state. Analysis of the non-heme iron content of HbRC cores indicates an antenna size of 21.6 +/- 1.1 BChl g molecules/P798. The evidence indicates that the HbRC contains a [4Fe-4S] cluster identified as F(X) that is coordinated between the PshA homodimer; in contrast to F(X) in other type I reaction centers, this [4Fe-4S] cluster exhibits an S = (3)/(2) ground spin state.