Time-resolved visible and infrared difference spectroscopy for the study of photosystem I with different quinones incorporated into the A1 binding site

Abstract

Room (298 K) and low (77K) temperature time-resolved visible and infrared difference spectroscopy has been used to study photosystem I particles with phylloquinone (2-methyl-3-phytyl-1,4-naphthoquinone), menadione (2-methyl-1,4-naphthoquinone) and plastoquinone 9 (2,3-dimethyl-5-prenyl-l,4-benzoquinone), incorporated into the A1 binding site. Concentrated samples in short path-length (~5 μm) sample cells are typically used in FTIR experiments. Measurements were undertaken using standard "dilute" samples at 298 K, and concentrated (~5×) samples at both 298 and 77K. No concentration induced alterations in the flash-induced absorption changes were observed. Concentrated samples in short path-length cells form a transparent film at 77K, and could therefore be studied spectroscopically at 77K without addition of a cryoprotectant. At 298 K, for photosystem I with plastoquinone 9/menadione/phylloquinone incorporated, P700+FA/B- radical pair recombination is characterized by a time constant of 3/14/80 ms, and forward electron transfer from A1A- to Fx by a time constant of 211/3.1/0.309 μs, respectively. At 77K, for concentrated photosystem I with menadione/phylloquinone incorporated, P700+A1- radical pair recombination is characterized by a time constant of 240/340 μs, with this process occurring in 58/39% of the PSI particles, respectively. The origin of these differences is discussed. Marcus electron transfer theory in combination with kinetic modeling is used to simulate the observed electron transfer time constants at 298 K. This simulation allows an estimate of the redox potential for the different quinones in the A1 binding site.

Modeling electron transfer in photosystem I

“…As indicated above, following light excitation the P700 + A 1  state forms in ~50 ps, and ET from A 1  to F X proceeds biphasically with time constants of 10-25 and 260-340 ns [16][17][18][19][20][21].…”

“…[21,25]. The experimentally observed rates can only be equated directly with k et when ET is largely exothermic and unidirectional, a situation that usually does not apply to ET reactions in PSI.…”

“…Inactivation of the menB gene inhibits the biosynthesis of PhQ, and it was subsequently found that plastoquinone-9 (PQ 9 ) is recruited into the A 1 binding site in PSI particles isolated from these so called menB null mutants (menB  PSI) [30,31]. By incubating menB  PSI with a large molar excess of the quinone of interest in vitro, the loosely-bound PQ 9 in the A 1 binding site is displaced by the non-native quinone [21,29,32,33]. This incubation method therefore provides an elegant approach for incorporating non-native…”

“…In previous time-resolved spectroscopy experiments we have obtained time constants associated with forward ET from A 1  to F X at RT in PSI with PhQ, 2MNQ and PQ 9 incorporated [21]. A basic kinetic model was considered (Figure 1, inset) and applied in combination with Equation 5 to estimate the midpoint potential for the different quinones in the A 1 binding site that was compatible with the experimentally observed rates of forward ET (from A 1  to F X at RT).…”

“…P700 + A 1  and P700 + F X  radical pair recombination reactions occur in 35-38% and 13-25% of the particles, respectively, while the P700 + F A/B  state is irreversibly formed in 40-49% of the PSI particles [16,21]. At 77 K, in cyanobacterial PSI particles from S6803 the P700 + A 1  state recombines with a lifetime (τ 1/e ) of ~340 μs [21], while the P700 + F X -states recombines in ~1-5 ms [16]. The fractional utilization of the B-branch is greatly reduced (~5%) at cryogenic temperature [22], and P700 + A 1  recombination occurs almost exclusively on the A-branch [29].…”