Primary acceptors Ao

The PSI-A and PSI-B polypeptides with molecular masses around 82 kDa (3,4,5) are homologous as demonstrated from nucleotide sequencing of their chloroplast genes psaA and psaB in a number of plant species (8-13). A heterodimer of the polypeptides are thought to bind the reaction center P700, the primary acceptors Ao, Ai and X, and about 60 chlorophyll a antennae molecules (4,5,6,29). This complex is often referred to as... 

Fig. 3. (A) Difference spectrum constructed from absorbance change transients in PS-1 particles poised at —625 mV [in the presence of dithionite] in a pH 10 buffer. Each transient was induced by a 3-s iliumination (B) AA obtained directiy with a P700-enriched, PS-i particie in a conventionai, commerciai spectrophotometer in the light-minus-dark mode (C) AA between 350 and 750 nm measured at 200 K with a Triton-fractionated, PS-1 particle from pea chloroplasts the spectrum shown was obtained by first illuminating at 200 K for 45 m and then at 215 K for three 30-m incremental periods. Figure source (A) Swarthoff, Gast, Amesz and Buisman (1982) Photoaccumulation of reduced primary electron acceptors of photosystem I of photosynthesis. FEBS Lett 146 131 (B) Ikegami and Ke (1984) A 160-kilodalton photosystem-l reaction-center complex. Low temperature absorption and EPR spectroscopy of the early electron acceptors. Biochim Biophys Acta 764 75 (C) Mansfield and Evans (1985) Optical difference spectrum of the electron acceptor Ao in photosystem I. FEBS Lett 190 239.

The lifetime of the charge-transfer state, [P700 Ao ], is expected to be extremely short and the time required to form this state shorter yet. Thus attempts to use picosecond spectroscopy to examine the behavior of Aq were made soon after the studies described above were reported. Following the earlier picosecond studies in 1979, additional studies began to provide not only more detailed but considerably improved spectral and kinetic information about the primary acceptor Aq. In this section, we will summarize the results of these picosecond studies in approximate chronological order. 

The difference spectrum for [Aq -Ao] shows a major bleaching at 690 nm plus a shoulder at 675 nm. Except for the 675-nm shoulder, the net spectrum obtained here also agrees with that obtained by Nuijs et using a 532-nm excitation pulse [cf. Fig. 6 (C)]. It also agrees with tbe in vitro spectrum for the Chl-a anion radical of Fujita et alf, except for a -25 nm red shift. Shuvalov et al thus concluded that the primary electron acceptor Ao in photosystem 1 is a Chi a-type molecule with a major absorption near 690 nm and has a forward electron-transfer time of 32 5 ps. In the pre-reduced state, the induced radical pair recombines in -55 ns, with partial conversion into triplet P700. 

When the level of excitation was kept low (-0.25 photon/RC), the overall decay of electronic excitation in the antenna was characterized by a time constant of 24-28p, from measurements of absorbance as well antenna fluorescence. The 24-28-ps process was observed in absorbance changes for P700 formation only, none for the reduction of the primary acceptor. This result confirms the previous finding of Holzwarth et al. and also supports their suggestion that the rate constant for Ao reoxidation is greater than the rate constant for its formation, which would make the absorbance changes hard to detect. 

Excitation energy is believed to be transferred to antenna pigments absorbing at the longest wavelength, BChl g-808, and subsequently to the reaction center [4-6]. The primary donor, P-798, in the reaction center is presumably a dimer of BChl g the 13 epimer of BChl g [7]. In the reaction center charge separation is induced between the primary donor and the primary acceptor A, identified as 8 -hydroxy Chi a [8]. When electron transport to the secondary acceptor(s) is blocked, P798 Ao recombines to the triplet state of P-798 with a yield of 30% at low temperatures [9]. The acceptor side of the heliobacterial reaction center seems to be similar to that of Photosystem I (PSI) of plants. It contains a Chi a derivative as primary acceptor and secondary electron transport involves at least one iron-sulfur cluster and possibly a quinone molecule [10-14]. However, there still remains substantial uncertainty about the order of electron transfer from the primary acceptor to the secondary acceptors. 

Melkozemov et d also measured the difference spectra for the reduction of the primary electron acceptor Aq, i.e., AA[Ao -Ao] in the wild-type and mutant PS-I cores placed under highly reducing conditions to maintain the secondary acceptors in a chemically reduced state, and found them to be identical, indicating that the primary electron acceptor Aq is not affected by PsaB His-656->Asn mutation. 

Note that the newly estimated lifetime of 32 5 ps for the photoreduced primary electron acceptor is shorter than the 200 ps reported previously This difference has been attributed to the different nature of the PS-I particles used in the two studies. As the PS-I particle used in the earlier studies had only 26 Chi per P700 v. a ratio of 75-100 in the particles used in the present studies, the extensive detergent treatment used to achieve the high P700-to-chlorophyll ratio might have affected the electron-transfer rate of Ao" and consequently its lifetime. The origin of the additional shoulder at 675 nm in the [Ao -Aq] difference spectrum has not yet been established it was tentatively explained by Sbuvalov et al. as possibly due to some contribution from antenna-cbloropbyll triplet state, in spite of tbe use of 710-nm excitation. 

Transient difference spectra ofthevitamin Kj-depleted and vitamin K3-reconstituted particle recorded at the appropriate time after the flash can provide another approach for obtaining the difference spectmm ofthe primary electron acceptor Aq of photosystem 1, as illustrated by Kim et al in Fig. 9 (B). Difference spectra ofthe vitamin Ki-depleted (solid line) and the vitamin K3-reconstituted particles (dotted line) were recorded 2 ns after the flash. The former spectmm should represent the formation of the radical pair, i.e., AA [P700 - P700] + [Ao -Aq] in the absence of a secondary acceptor. The latter... 

Fig. 10. (A) Absorbance-difference spectra measured at various times foliowing excitation of a PS ll-deieted mutant of Synechocystis sp. by l50-fe, 590-nm pulses. Spectra obtained from open [O] and closed [ ) reaction centers are shown in panels (a) and (d). respectively panel (b) shows the expanded open- and closed-difference spectra at 89 ps panel (c) shows the open-minus-closed difference spectrum, /.e., A[aa], at 89 ps panei (e) shows simiiar A[AA]s at deiay times of 1,10, and 25 ps. (B), panel (a) shows "decay-associated difference spectra" (DADS) of the 4-, 21-ps and the nondecaying component obtained by global analysis of data from the open and closed reaction centers shown in (A) (B, b) is a comparison of the 21-ps DADS with the nanosecond [reduced-minus-open] AA[Ao"-Ao] (B, c) is a comparison of the nondecaying DADS with the AA[P700 -P700] measured on the microsecond time scale. Data source Hastings, Kleinherenbrink, Lin, McHugh and Blankenship (1994) Observation of the reduction and reoxidation of the primary eiectron acceptor in photosystem i. Biochemistry 33 3196, 3197.

Fig. 12. Transient difference spectra induced by 1-ps flash in PS-I particles with a Chl/P700=30. Spectra for particles with the native phylloquinone extracted (-Q) are shown in panel (A) and those for particles reconstituted with menaquinone-4 (+Q) are shown in panel (A )- Spectra for particles with open (O) and closed ( ) reaction centers are drawn as solid and dotted lines, respectively. For the quinone-depleted and quinone-reconstituted particles, difference spectra were recorded in panels (B) and (B ) at three delay times, as indicated. Excitation intensity corresponds to 1.5 photons absorbed per RC. Panels (C) and (C ) show plots of A[AA]s at 695 and 685 nm vs. various delay times after the excitation flash, showing the rise and decay kinetics of P700 and Ao, respectively. Figure source Kumazaki, Iwaki, Ikegami, Kandori, Yoshihara and Itoh (1994) Rates of primary electron transfer reactions in the photosystem I reaction center reconstituted with different quinones as the secondary acceptor. J Phys Chem 98 11221,11222.

The reducing side of photosystem I (PS I) reaction center of oxygenic photosynthetic organisms is known to consist of five different acceptors (1,2). The primary electron acceptor, called AO, is assumed to be a monomeric chlorophyll molecule. An electron is passed from AO to a secondary acceptor A1 which is believed to be phylloquinone. The electron transfer involves three different iron-sulfur centers, called FX, FB and FA. PS I high-molecular-mass subunits (about 82 kDa) are known to contain AO, Al, and FX as well as P700 (1,2). FA and FB are believed to be located in a small polypeptide of about 9 kDa (3,4). As we demonstrated elsewhere (5), heat treatment of spinach PS I particles in the presence of ethylene glycol (EG) caused the selective destruction of the iron-sulfur centers and led to the dissociation of polypeptides from the particles. A small subunit of about 5 kDa was closely associated with large subunits under this treatment. In this paper, we present the N-terminal amino acid sequence of the 5 kDa polypeptide. 

P700, the primary electron donor of PSI, and Ao, the electron acceptor, are chlorophylls, P700 having a redox potential of around -490 mV. Ai is possibly a quinone, and FeSx, FeSA, and FeSe are iron-sulfur centers with potentials of -705, -530, and -580 mV, respectively. FeSA and FeSe probably operate in parallel and denote electrons to ferredoxin (redox potential, -420 mV) (Figure 1.7). 

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