Photosystem I (PS I) contains two molecules of phylloquinone that function as electron transfer cofactors at highly reducing midpoint potentials. It is therefore surprising that each phylloquinone is hydrogen bonded at the C 4 position to the backbone -NH of a Leu residue since this serves to drive the midpoint potential more oxidizing. To better understand the role of the H-bond, a PS I variant was generated in which L722 PsaA was replaced with a bulky Trp residue. This change was designed to alter the conformation of the A-jk(1) loop and therefore change the strength of the H-bond to the PsaA-branch phylloquinone. Transient EPR studies at 80 K show that the A 1A site in the PS I variant is fully occupied with phylloquinone, but the absence of methyl hyperfine couplings in the quinone contribution to the P 700 +A 1 - radical pair spectrum indicates that the H-bond has been weakened. In wild-type PS I, reduction of F A and F B with sodium dithionite causes a ∼30% increase in the amplitude of the P 700 +A 1 - transient EPR signal due to the added contribution of the PsaB-branch cofactors to low temperature reversible electron transfer between P 700 and A 1A. In contrast, the same treatment to the L722W PsaA variant leads to a ∼75% reduction in the amplitude of the P 700 .+A 1 .- transient EPR signal. This behavior suggests that A 1A has undergone double reduction to phyllohydroquinone, thereby preventing electron transfer past A 0A. The remaining 25% of the P 700 .+A 1 .- radical pair spectrum shows an altered spin polarization pattern and pronounced methyl hyperfine couplings characteristic of asymmetric H-bonding to the phylloquinone. Numerical simulations of the polarization pattern indicate that it arises primarily from electron transfer between P 700 and A 1B. The altered reduction behavior in the L722W PsaA variant suggests that the primary purpose of the H-bond is to tie up the C 4 carbonyl group of phylloquinone in a H-bond so as to prevent protonation and hence lower the probability of double reduction during periods of high light intensity.
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