The issue of whether one or both branches of electron-transfer cofactors is active in Photosystem I (PS I) was studied using a strategy employing interposon mutagenesis and electron paramagnetic resonance (EPR) spectroscopy. PS I complexes were isolated using n-dodecyl-beta-D-maltoside (DM) or Triton X-100 (TX-100) from wild-type and mutant strains of Synechococcus sp. PCC 7002 lacking specific PS I polypeptides. The principal values of the g-tensor of Al were determined by Q-band (34.0 GHz) EPR spectroscopy of perdeuterated, wild-type PS I complexes as g(xx) = 2.0062, g(yy) = 2.0050, and g(zz) = 2.0021, and a stoichiometry of less than or equal to 1.0 A(1)(-) per P700(+) was measured by spin quantitation of photoaccumulated, wild-type PS I complexes at illumination temperatures between 195 and 220 K. The characteristic anisotropic EPR spectrum of A(1)(-) was photoaccumulated in all mutant PS I complexes isolated with DM and most mutant PS I complexes isolated with TX-100; however, A(0)(-) was photoaccumulated in PS I complexes isolated with TX-100 from the psaF and psaE psaF mutants. PS I complexes isolated with TX-100 from the wild type and the psaE psaF mutant retained 2.5 phylloquinone (PhQ) and 2.6 PhQ per 100 Chi, respectively, indicating that the failure to observe A(1)(-) in the psaE psaF mutant is not due to the loss of PhQ. Steady-state rates of light-induced flavodoxin reduction for PS I complexes isolated with DM and TX-100 from the psaF and psaE psaF mutants were nearly identical and differed by less than a factor of 2 from that for the wild-type. The inability to photoaccumulate a second A(1)(-) in the wild-type and the psaE psaF mutant indicates that only one of the two resident PhQs in PS I is redox active. The presence of A(0)(-) in the psaE and psaE psaF mutants is explained by the protonation and secondary reduction of the semiquinone anion radical of PhQ which is made solvent-accessible by removal of the PsaF polypeptide and by exposure to TX-100. Since only the loss of the C-3-distal PsaF or PsaE and PsaF polypeptides affects the spectroscopic properties of A(1), the binding site of the redox-active PhQ is associated with the nonprimed cr-helices assigned to PsaA/PsaB the electron density map.