We created a Qo pocket mutant by site-directed mutagenesis of the chloropla
st petD gene in Chlamydomonas reinhardtii. We mutated the conserved PEWY se
quence in the EF loop of subunit IV into PWYE. The pwye mutant did not grow
in phototrophic conditions although it assembled wild-type levels of cytoc
hrome b(6)f complexes. We demonstrated a complete block in electron transfe
r through the cytochrome b(6)f complex and a loss of plastoquinol binding a
t Qo, The accumulation of cytochrome b(6)f complexes lacking affinity for p
lastoquinol enabled us to investigate the role of plastoquinol binding at Q
o in the activation of the light-harvesting complex II (LHCII) kinase durin
g state transitions. We detected no fluorescence quenching at room temperat
ure in state II conditions relative to that in state I. The quantum yield s
pectrum of photosystem I charge separation in the two state conditions disp
layed a trough in the absorption region of the major chlorophyll a/b protei
ns, demonstrating that the cells remained locked in state I. P-33(i) labeli
ng of the phosphoproteins in vivo demonstrated that the antenna proteins re
mained poorly phosphorylated in both state conditions. Thus, the absence of
state transitions in the pwye mutant demonstrates directly that plastoquin
ol binding in the Qo pocket is required for LHCII kinase activation.