<p>Early processes following photon absorption by the photosynthetic pigment-protein complex photosystem I (PS I) have been the subject of decades of research, yet many questions remain in this area of study. Among the trickiest to investigate is the role of the PS I reaction center’s (RC’s) two accessory (A_‑1) chlorophyll (Chl) cofactors as primary electron donors or acceptors, oxidizing the special pair (P₇₀₀) of Chls or reducing a nominal primary electron acceptor (A₀) Chl in the first electron transfer step. Such processes, which occur on a picosecond timescale, have long been studied via ultrafast spectroscopy, though difficulty lies in distinguishing among signals from early processes, which have similar lifetimes and involve many identical pigments. In this work, we used steady-state and ultrafast optical pump-probe spectroscopies on PS I trimers from wildtype and mutant strains of the cyanobacterium <i>Synechocystis</i> sp. PCC 6803 in which an asparagine amino acid residue near A_‑1 had been replaced with methionine on one or both sides of the RC. We also conducted an identical set of experiments on mutants in which A₀ was similarly targeted, as well as studied the effects on the A₀ absorption spectrum of a third category of mutations in which a peripheral H‑bond to A₀ was lost. Steady-state absorption spectroscopy revealed that many of these mutations caused mild Chl deficiencies in the light-capturing antenna of PS I without necessarily preventing organisms’ growth. More importantly, we determined that contrary to certain hypotheses, A_‑1 is the most likely true first electron acceptor, as reasoned from observing rapid triplet state formation in double A_‑1 mutants. We also concluded from non-additive detrimental effects of single-side mutations that if one RC branch is damaged at the level of A₀ or A_‑1, electron transfer may be redirected along the intact branch. This may help explain the conservation of two functional RC branches in PS I over many generations of natural selection, despite the additional cost to organisms of manufacturing both.</p>