Factors controlling the redox properties of the two conventional forms of c
ytochrome b559, i.e. the unstable high-potential form and the stable low-po
tential form, have been further investigated using PSII-enriched membranes
from pea and spinach chloroplasts. The redox potential of the stable form o
f cytochrome b559 is pH independent both above pH 7.5 (E'(m) approximate to
+110 mV) and below pH 6.0 (E'(m) approximate to +203 mV), but it changes w
ith a slope of 58 mV per pH unit between these two pH values. Thus, cytochr
ome b559 seems to have a single ionizing group influencing its redox potent
ial, with a higher affinity for protons in the reduced form (pK(red) = 7.5)
and a lower affinity in the oxidized form (pK(ox) = 6.0); consequently, on
e unprotonated low-potential form (LP) and one protonated intermediate-pote
ntial form (IP). The redox potential of the high-potential form (HP) is pH-
independent between pH 5.0 and 8.0, but its relative content (compared to t
he total amount of protein) decreases progressively above pH 7.0. This conv
ersion to the stable LP form is interpreted as corresponding to the loss of
a proton by one ionizing group, the protonation of which is essential for
maintaining the unstable HP state. According to chemical modification exper
iments with diethylpyrocarbonate, one of the two histidine ligands of the h
eme seems to be the ionizing group responsible for the existence of both th
e protonated EP and HP forms. It is proposed that the difference between th
e EP and HP forms is due to the formation of an additional hydrogen bond be
tween the protonated histidine and the protein in the HP state that stabili
zes a special hydrophobic heme environment responsible for its high redox p
otential.