Why is it so difficult to construct Q(i) site mutants in Chlamydomonas reinhardtii?

The cytochrome b(6)f complex catalyses electron transfer from plastoquinol to a hydrosoluble acceptor (plastocyanin), while building up an electrochemical proton gradient. Oxidation and reduction of plastoquinol occur respectively at the Q(o) site (exposed on the luminal side of the thylakoid membrane) and at the Q(i) site (facing the stroma). The discovery of an additional c'-type heme in the Q(i) site has cast a new light on the difficulties previously encountered to obtain mutants at this site. In this work, we critically examine our unsuccessful attempts to obtain Q(i) site mutants based on sequence and structure homology between cytochrome b(6)f and bc(1) complexes.