Recruitment of a Foreign Quinone into the A1 Site of Photosystem I

Abstract

Interruption of the menA or menB gene in Synechocystis sp. PCC 6803 results in the incorporation of a foreign quinone, termed Q, into the A 1 site of photosystem I with a number of experimental indicators identifying Q as plastoquinone-9. A global multiexponential analysis of time-resolved optical spectra in the blue region shows the following three kinetic components: 1) a 3-ms lifetime in the absence of methyl viologen that represents charge recombination between P700؉ and an FeS ؊ cluster; 2) a 750-s lifetime that represents electron donation from an FeS ؊ cluster to methyl viologen; and 3) an ϳ15-s lifetime that represents an electrochromic shift of a carotenoid pigment. Room temperature direct detection transient EPR studies of forward electron transfer show a spectrum of P700 ؉ Q ؊ during the lifetime of the spin polarization and give no evidence of a significant population of P700 ؉ FeS ؊ for t < 2-3 s. The UV difference spectrum measured 5 s after a flash shows a maximum at 315 nm, a crossover at 280 nm, and a minimum at 255 nm as well as a shoulder at 290 -295 nm, all of which are characteristic of the plastoquinone-9 anion radical. Kinetic measurements that monitor Q at 315 nm show a major phase of forward electron transfer to the FeS clusters with a lifetime of ϳ15 s, which matches the electrochromic shift at 485 nm of the carotenoid, as well as an minor phase with a lifetime of ϳ250 s. Electrometric measurements show similar biphasic kinetics. The slower kinetic phase can be detected using timeresolved EPR spectroscopy and has a spectrum characteristic of a semiquinone anion radical. We estimate the redox potential of plastoquinone-9 in the A 1 site to be more oxidizing than phylloquinone so that electron transfer from Q ؊ to F X is thermodynamically unfavorable in the menA and menB mutants.The overall goal of these studies is to replace phylloquinone (vitamin K 1 ; 2-methyl-3-phytyl-1,4-naphthoquinone) in the A 1 site of photosystem I with a foreign quinone of different redox and/or kinetic properties but still capable of forward electron transfer to the FeS 1 clusters. In two previous papers, we described the construction and physiology of phylloquinone biosynthetic mutants in Synechocystis sp. PCC 6803 (1), and we reported EPR and electron nuclear double resonance studies that implied the presence of a foreign quinone, termed Q, in the A 1 site of PS I (2). To summarize briefly, the interruption of the menA and menB genes, which code for dihydroxynaphthoic acid synthase and phytyltransferase, respectively, results in the termination of phylloquinone biosynthesis as judged by high pressure liquid chromatography/mass spectroscopy (HPLC/ MS) and gas chromatography/mass spectroscopy of pigment extracts from isolated PS I complexes. However, the menA and menB mutant strains grow photoautotrophically under low light intensities, and isolated PS I complexes are capable of sustaining high rates of steady-state electron transfer from cytochrome c 6 to flavodoxin. HPLC and HPLC/MS studies show that quantita...

Redox Potential of Quinones in Both Electron Transfer Branches of Photosystem I

“…The lifetime of the reduced state A 1 Ϫ was measured to be about 1/k ET ϭ 280 ns (6, 58 -61). The measured values of the redox potential of F X vary between Ϫ705 and Ϫ650 mV (62)(63)(64)(65). Using rate expression (Equation 2) that is valid for exergonic ET processes with these parameters yields a redox potential for A 1 of Ϫ497 and Ϫ442 mV that is more positive than the value for F X .…”

Recruitment of a Foreign Quinone into the A1 Site of Photosystem I

“…For the wild type, a distance of 25.3 Ϯ 0.3 Å has been evaluated, which does not change for any of the substituted quinones investigated in this study. P700 ϩ Optical Recombination Kinetics-As we showed in our previous paper (15), the replacement of PhQ by PQ-9 results in altered kinetics of the backreaction from [F A /F B ]…”

“…15. To assure resolution of kinetics in the microsecond time domain, a high frequency roll-off amplifier described in the original specifications was not employed, and the signal was fed directly into the plug-in (11A33 differential comparator, 100-MHz bandwidth) of the Tektronix DSA601 oscilloscope.…”

“…In a series of three papers (13)(14)(15), we reported the generation and characterization of PhQ-less mutants in the cyanobacterium Synechocystis sp. PCC 6803.…”

Loss of Phylloquinone in Chlamydomonas Affects Plastoquinone Pool Size and Photosystem II Synthesis

“…Loss of phylloquinone synthesis has different effects in cyanobacteria and land plants. In cyanobacteria, insertional inactivation of the menA, -B, -D, and -E genes leads to the complete absence of phylloquinone, which is replaced by plastoquinone-9 in PSI (2,3,16,36). However, inactivation of menG, the last enzyme involved in the phylloquinone synthesis pathway, leads to the accumulation of demethylphylloquinone, which replaces phylloquinone in PSI (5).…”