Oxidoreductases of the cytochrome bc(1)/b(6)f family transfer electrons from a liposoluble quinol to a soluble acceptor protein and contribute to the formation of a transmembrane electrochemical potential. The crystal structure of cyt b(6)f has revealed the presence in the Q(i) site of an atypical c-type heme, heme c(i). Surprisingly, the protein does not provide any axial ligand to the iron of this heme, and its surrounding structure suggests it can be accessed by exogenous ligand. In this work we describe a mutagenesis approach aimed at characterizing the c(i) heme and its interaction with the Q(i) site environment. We engineered a mutant of Chlamydomonas reinhardtii in which Phe(40) from subunit IV was substituted by a tyrosine. This results in a dramatic slowing down of the reoxidation of the b hemes under single flash excitation, suggesting hindered accessibility of the heme to its quinone substrate. This modified accessibility likely originates from the ligation of the heme iron by the phenol(ate) side chain introduced by the mutation. Indeed, it also results in a marked downshift of the c(i) heme midpoint potential (from +100 mV to -200 mV at pH 7). Yet the overall turnover rate of the mutant cytochrome b(6)f complex under continuous illumination was found similar to the wild type one, both in vitro and in vivo. We propose that, in the mutant, a change in the ligation state of the heme upon its reduction could act as a redox switch that would control the accessibility of the substrate to the heme and trigger the catalysis.