Asymmetric Hydrogen-Bonding of the Quinone Cofactor in Photosystem I Probed by 13C-Labeled Naphthoquinones

In photosystem I (PS I) the phylloquinone secondary acceptor functions as a one electron gate similar to the Q A quinone secondary acceptor in type II reaction centers. The quinone radical anion formed during charge separation as part of the P 700 +• A 1 -• radical pair state is known to have a highly asymmetric electron spin density distribution, which is attributed to asymmetric hydrogen bonding with the protein environment. Here, the native phylloquinone is replaced by specifically 13 C-labeled 2-methyl-1,4-naphthoquinones (2-methyl-4- 13 C-1,4-naphthoquinone and 2- 13 C-methyl-1,4-naphthoquinone) to probe the spin density distribution in two relevant quinone ring positions. The men B phylloquinone biosynthetic pathway mutant was used because it allows for efficient in vitro quinone replacement in isolated PS I trimers. X- and Q-band time-resolved EPR spectroscopy was used to determine the 13 C hyperfine tensors in the functional P 700 +• A 1 -• state. For 2-methyl-4- 13 C-1,4-naphthoquinone anion radical the largest hyperfine tensor component A zz = 44 MHz was found to be considerably larger than those determined in bacterial reaction centers. The PS I structure at 2.5 Å resolution shows a single H-bond from the backbone NH group of a specific leucine residue. Such a highly asymmetric H-bonding is confirmed here for the functional P 700 +• A 1 -• state. Experimental evidence for an unusual backbone H-bond strength is analyzed by comparing the quinone binding site with that in bacterial reaction centers and possible mechanisms of increased H-bond strength are considered.