Recruitment of a foreign quinone into the A₁ site of photosystem I. II. Structural and functional characterization of phylloquinone biosynthetic pathway mutants by electron paramagnetic resonance and electron-nuclear double resonance spectroscopy

Electron paramagnetic resonance (EPR) and electron-nuclear double resonance studies of the photosystem (PS) I quinone acceptor, A₁, in phylloquinone biosynthetic pathway mutants are described. Room temperature continuous wave EPR measurements at X-band of whole cells of menA and menB interruption mutants show a transient reduction and oxidation of an organic radical with a g-value and anisotropy characteristic of a quinone. In PS I complexes, the continuous wave EPR spectrum of the photoaccumulated Q^- radical, measured at Q-band, and the electron spin-polarized transient EPR spectra of the radical pair P700⁺ Q^-, measured at X-, Q-, and W-bands, show three prominent features: (i) Q^- has a larger g-anisotropy than native phylloquinone, (ii) Q^- does not display the prominent methyl hyperfine couplings attributed to the 2-methyl group of phylloquinone, and (iii) the orientation of Q^- in the A₁ site as derived from the spin polarization is that of native phylloquinone in the wild type. Electron spin echo modulation experiments on P700⁺ Q^- show that the dipolar coupling in the radical pair is the same as in native PS I, i.e. the distance between P700⁺ and Q^-(25.3 ± 0.3 Å) is the same as between P700⁺ and A₁^- in the wild type. Pulsed electron-nuclear double resonance studies show two sets of resolved spectral features with nearly axially symmetric hyperfine couplings. They are tentatively assigned to the two methyl groups of the recruited plastoquinone- 9, and their difference indicates a strong inequivalence among the two groups when in the A₁ site. These results show that Q (i) functions in accepting an electron from A₀^- and in passing the electron forward to the iron-sulfur clusters, (ii) occupies the A₁ site with an orientation similar to that of phylloquinone in the wild type, and (iii) has spectroscopic properties consistent with its identity as plastoquinone-9.