A gene encoding a methyltransferase (menG) was identified in Synechocystis sp. PCC 6803 as responsible for transferring the methyl group to 2-phytyl-1,4-naphthoquinone in the biosynthetic pathway of phylloquinone, the secondary electron acceptor in photosystem I (PS I). Mass spectrometric measurements showed that targeted inactivation of the menG gene prevented the methylation step in the synthesis of phylloquinone and led to the accumulation of 2-phytyl-1,4-naphthoquinone in PS I. Growth rates of the wild-type and the menG mutant strains under photoautotrophic and photomixotrophic conditions were virtually identical. The chlorophyll a content of the menG mutant strain was similar to that of wild type when the cells were grown at a light intensity of 50 microE m(-2) s(-1) but was slightly lower when grown at 300 microE m(-2) s(-1). Chlorophyll fluorescence emission measurements at 77 K showed a larger increase in the ratio of PS II to PS I in the menG mutant strain relative to the wild type as the light intensity was elevated from 50 to 300 microE m(-2) s(-1). CW EPR studies at 34 GHz and transient EPR studies at multiple frequencies showed that the quinone radical in the menG mutant has a similar overall line width as that for the wild type, but consistent with the presence of an aromatic proton at ring position 2, the pattern of hyperfine splittings showed two lines in the low-field region. The spin polarization pattern indicated that 2-phytyl-1,4-naphthoquinone is in the same orientation as phylloquinone, and out-of-phase, spin-echo modulation spectroscopy shows the same P700(+) to Q(-) center-to-center distance as in wild-type PS I. Transient EPR studies indicated that the lifetime for forward electron transfer from Q(-) to F(X) is slowed from 290 ns in the wild type to 600 ns in the menG mutant. The redox potential of 2-phytyl-1,4-naphthoquinone is estimated to be 50 to 60 mV more oxidizing than phylloquinone in the A(1) site, which translates to a lowering of the equilibrium constant between Q(-)/Q and F(X)(-)/F(X) by a factor of ca. 10. The lifetime of the P700(+) [F(A)/F(B)](-) backreaction decreased from 80 ms in the wild type to 20 ms in the menG mutant strain and is evidence for a thermally activated, uphill electron transfer through the quinone rather than a direct charge recombination between [F(A)/F(B)](-) and P700(+).