Effects of carboxyl amino acid modification on the properties of the high-affinity, manganese-binding site in photosystem II.
Our previous work using the "diphenylcarbazide (DPC)-inhibition assay" has identified four amino acid (two carboxyls and two histidyls) ligands 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 al. (1996) Biochemistry 35, 1820-1828]. The current paper shows the following: (a) the high-affinity photooxidizable Mn, which donates to the oxidized primary PSII donor (YZ*), is bound to a carboxyl residue with a KM = 1.5 microM or Kd = 0.94 microM in the absence of DPC, and a Ki = 1.3 microM in the presence of DPC (both steady-state and flash approaches were used); (b) if this carboxyl is chemically modified using 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride (EDC), Mn2+ is photooxidized at a lower affinity (Kd = 25 microM) site that does not involve carboxyl ligands; (c) low-affinity Mn is photooxidized (possibly by YD*, the oxidized form of the alternative PSII donor) with a KM = 220 microM at a completely different site that also requires a carboxyl ligand; (d) photooxidation of high-affinity DPC by YZ* with a KM of 40-42 microM or Kd of 49-58 microM occurs at a site that does not require carboxyl residues; (e) photooxidation of low-affinity DPC with a KM = 1200 microM occurs at a site (possibly near YD) that is not affected by carboxyl modification with EDC. Due to the similarities between the binding of the high-affinity photooxidizable Mn to EDC-treated membranes and to PSII complexes from Asp170D1 mutants [Nixon and Diner (1992) Biochemistry 31, 942-948], we identify its carboxyl residue ligand as Asp170 on D1, one of the reaction-center proteins. The second carboxyl ligand identified using the DPC-inhibition assay binds Mn (but not a photooxidizable one), Zn, 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+.
Ghirardi
M L
ML
National Renewable Energy Laboratory, Golden, Colorado 80401, USA.
Lutton
T W
TW
Seibert
M
M
D015086:2,6-Dichloroindophenol / Q000378:metabolism; D019943:Amino Acid Substitution; D001224:Aspartic Acid / Q000378:metabolism; D001665:Binding Sites; D004160:Diphenylcarbazide / Q000037:antagonists & inhibitors / Q000378:metabolism; D004237:Diuron / Q000378:metabolism; D005022:Ethyldimethylaminopropyl Carbodiimide / Q000494:pharmacology; D008024:Ligands; D008345:Manganese / Q000737:chemistry* / Q000378:metabolism*; D010084:Oxidation-Reduction; D010777:Photochemistry; D045322:Photosynthetic Reaction Center Complex Proteins / Q000737:chemistry* / Q000378:metabolism*; D045332:Photosystem II Protein Complex; D013050:Spectrometry, Fluorescence; D018724:Spinacia oleracea
D008024:Ligands; D045322:Photosynthetic Reaction Center Complex Proteins; D045332:Photosystem II Protein Complex; D004160:Diphenylcarbazide; D001224:Aspartic Acid; D008345:Manganese; D004237:Diuron; D015086:2,6-Dichloroindophenol; D005022:Ethyldimethylaminopropyl Carbodiimide