Photosystem I (PS I) has two nearly identical branches of electron-transfer
co-factors. Based on point mutation studies, there is general agreement that
both branches are active at ambient temperature but that the majority of
electron-transfer events occur in the A-branch. At low temperature, reversible
electron transfer between P700 and A1A occurs in the A-branch. However, it has
been postulated that irreversible electron transfer from P700 through A1B to
the terminal iron-sulfur clusters FA and FB occurs via the B-branch. Thus, to
study the directionality of electron transfer at low temperature, electron
transfer to the iron-sulfur clusters must be blocked. Because the geometries
of the donor–acceptor radical pairs formed by electron transfer in the A- and
B-branch differ, they have different spin-polarized EPR spectra and echo-
modulation decay curves. Hence, time-resolved, multiple-frequency EPR
spectroscopy, both in the direct-detection and pulse mode, can be used to
probe the use of the two branches if electron transfer to the iron-sulfur
clusters is blocked. Here, we use the PS I variant from the menB deletion
mutant strain of Synechocyctis sp. PCC 6803, which is unable to synthesize
phylloquinone, to incorporate 2,3-dichloro-1,4-naphthoquinone (Cl2NQ) into the
A1A and A1B binding sites. The reduction midpoint potential of Cl2NQ is
approximately 400 mV more positive than that of phylloquinone and is unable to
transfer electrons to the iron-sulfur clusters. In contrast to previous
studies, in which the iron-sulfur clusters were chemically reduced and/or
point mutations were used to prevent electron transfer past the quinones, we
find no evidence for radical-pair formation in the B-branch. The implications
of this result for the directionality of electron transfer in PS I are
discussed