A photosystem I (PS I) complex containing plastoquinone-9 (PQ-9) but devoid of F(X), F(B), and F(A) was isolated and characterized from a mutant strain of Synechococcus sp. PCC 7002 in which the menB and rubA genes were insertionally inactivated. In isolated PS I trimers, the decay of P700+ measured in the near-IR and the decay of A1- measured in the near-UV were found to be biphasic, with (averaged) room temperature lifetimes of 12 and 350 micros. The decay-associated spectra of both kinetic phases are characteristic of the oxidized minus reduced difference spectrum of a semiquinone, consistent with charge recombination between P700+ and PQ-9-. The amplitude of the flash-induced absorbance changes in both the near-IR and the near-UV show that approximately one-half of the A1 binding sites are either empty or nonfunctional. A spin-polarized chlorophyll triplet is observed by time-resolved EPR, and it is attributed to the 3P700 product of P700+A0- charge recombination via the T0 spin level in those PS I complexes that do not contain a functional quinone. In those A1 sites that are occupied, the P700+Q- polarization pattern indicates that PQ-9 is oriented in a similar manner to that in the menB mutant. When excess 9,10-anthraquinone is added in vitro, it displaces PQ-9 and occupies the A1 binding site more readily than in the menB mutant. This can be explained by a greater accessibility to the A1 site in the menB rubA mutant due to the absence of F(X) and the stromal ridge polypeptides. The relatively low binding affinity of 9,10-anthraquinone allows it to be readily removed from the A1 site by washing. However, all A1 sites are shown to bind napthoquinones with high affinity and thus are proven to be functionally competent in quinone binding. The ability to readily displace PQ-9 from the A1 site makes the menB rubA mutant ideal for introducing novel quinones, particularly anthraquinones, into PS I.