Ml. Ghirardi et al., EFFECTS OF CARBOXYL AMINO-ACID MODIFICATION ON THE PROPERTIES OF THE HIGH-AFFINITY, MANGANESE-BINDING SITE IN PHOTOSYSTEM-II, Biochemistry (Easton), 37(39), 1998, pp. 13559-13566
Our previous work using the ''diphenylcarbazide (DPC)-inhibition assay
'' has identified-four amino acid (two carboxyls and two histidyls) li
gands to four Mn2+ bound with high affinity on Tris-washed photosystem
II (PSII) membrane fragments [Preston and Seibert (1991) Biochemistry
30, 9615-9624, 9625-9633]. One of the ligands binds a photooxidizable
Mn, specifically, and the others bind either nonphotooxidizable Mn2+,
Zn2+, or Co2+ [Ghirardi et all (1996) Biochemistry 35, 1820-1828]. Th
e current paper shows the following: (a) the high-affinity photooxidiz
able Mn, which donates to the oxidized primary PSII donor (Y-z(.)), is
bound to a carboxyl residue with a K-M = 1.5 mu M or K-d = 0.44 mu M
in the absence of DPC, and a K-i = 1.3 mu M in the presence of DPC (bo
th steady-state and flash approaches were used); (b) if this carboxyl
is chemically modified using 1-ethyl-3-[3-(dimethylamino)propyl] carbo
diimide hydrochloride (EDC), Mn2+ is photooxidized at a lower affinity
(K-d = 25 mu M) site that does not involve carboxyl ligands; (c) low-
affinity Mn is photooxidized (possibly by Y-D(.), the oxidized form of
the alternative PSII donor) with a K-M = 220 mu M at a completely dif
ferent site that also requires a carboxyl ligand; (d) photooxidation o
f high-affinity DPC by Y-Z(.) with a K-M of 40-42 mu M or K-d of 49-58
mu M occurs at a site that does not require carboxyl residues; (e) ph
otooxidation of low-affinity DPC with a K-M = 1200 mu M occurs at a si
te (possibly near Y-D) that is not affected by carboxyl modification w
ith EDC. Due to the similarities between the binding of the high-affin
ity photooxidizable Mn to EDC-treated membranes and to PSII complexes
from Asp 170D1 mutants [Nixon and Diner (1992) Biochemistry 31, 942-94
8], we identify its carboxyl residue ligand as Asp170 on D1, one of th
e reaction-center proteins. The second carboxyl ligand identified usin
g the DPC-inhibition assay binds Mn (but not a photooxidizable one), Z
n, or Co ions. At least one of the two histidyl ligands (either His337
on D1 or another unidentified histidyl) that bind nonphotooxidizable,
high-affinity Mn2+ also binds Zn2+ and Co2+.