Role of the Hydrogen Bond from Leu722 to the A1A Phylloquinone in Photosystem I

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)(center dot+)A(1)(center dot-) 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 similar to 30% increase in the amplitude of the P(700)(center dot+)A(1)(center dot-) 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 similar to 75% reduction in the amplitude of the P(700)(center dot+)A(1)(center dot-) 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)(center dot+)A(1)(center dot-) 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 P700 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.