In the cyanobacterium Synechocystis sp. PCC 6803, photosynthetic electron transport from the cytochrome bf complex to photosystem I can be mediated by cytochrome c553 or plastocyanin. The concentration of copper in the growth medium determines which protein is synthesized. To investigate the role of cytochrome c553 in photosynthetic and respiratory electron transport, we cloned the petJ gene encoding cytochrome c553 from Synechocystis 6803 and determined its nucleotide sequence. The 360-base pair open reading frame encodes an 85-amino acid mature protein (predicted molecular mass = 8,742 Da) with a 35-amino acid presequence. Two mutants were constructed, one in which the petJ gene for cytochrome c553 was deleted and another in which the petE gene for plastocyanin was insertionally inactivated. The cytochrome c553 deletion mutant (M109) grew photoautotrophically, even in the absence of copper that prevented the synthesis of plastocyanin, as did the plastocyanin-deficient mutant (M114) grown in the presence of copper that prevented the synthesis of cytochrome c553. The M109 strain exhibited photosynthetic electron transport rates similar to those of wild-type cells when grown under conditions that prevented the synthesis of plastocyanin. Moreover, in M109 cells grown without copper, cytochrome f was completely photooxidized in less than 10 ms by photosystem I. These observations show that electrons can be transferred from the cytochrome bf complex to photosystem I in the absence of both cytochrome c553 and plastocyanin. Additionally, the M109 cells exhibited dark respiration rates comparable with those of wild-type cells, indicating that cytochrome c553 is not obligately required for respiratory electron transport in Synechocystis 6803.