The catalytic role of subunit IV of the cytochrome b6-f complex from spinach chloroplast.
The catalytic role of subunit IV, the Mr 17,000 protein, in the chloroplast cytochrome b6-f complex was established through trypsinolysis of the complex under controlled conditions. When purified chloroplast cytochrome b6-f complex, 1 mg/ml, in 50 mM Tris-succinate buffer (pH 7.0) containing 1% sodium cholate and 10% glycerol is treated with 80 micrograms of trypsin at room temperature for various lengths of time, the activity of the cytochrome b6-f complex decreases as the incubation time increases. A maximal inactivation of 80% is reached at 7 min of incubation. The trypsin inactivation is accompanied by the destruction of the proton translocation activity of the complex. No alteration of absorption and EPR spectral properties was observed in the trypsin-inactivated complex. Subunit IV is the only subunit in the cytochrome b6-f complex that is digested by trypsin, and the degree of digestion correlates with the decrease of electron transfer activity. The binding of azido-Q to subunit IV of the complex decreases as the extent of inactivation of the cytochrome b6-f complex by trypsin increases. The residue molecular mass of trypsin cleaved subunit IV is about 14 kDa, suggesting that the cleavage site is at lysine 119 or arginine 125 or 126. When the thylakoid membrane was assayed for cytochrome b6-f complex activity, very little activity was observed; and the activity was not sensitive to trypsinolysis. Upon sonication, activity and sensitivity to trypsinolysis was greatly increased, suggesting that subunit IV protrudes from the lumen side of the membrane.
Li
L B
LB
Department of Biochemistry, OAES, Oklahoma State University, Stillwater 74078.
Zou
Y P
YP
Yu
L
L
Yu
C A
CA
D000345:Affinity Labels; D000595:Amino Acid Sequence; D015153:Blotting, Western; D002384:Catalysis; D002736:Chloroplasts / Q000201:enzymology*; D003573:Cytochrome b Group / Q000737:chemistry*; D045346:Cytochrome b6f Complex; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D010944:Plants; D014357:Trypsin
D000345:Affinity Labels; D003573:Cytochrome b Group; D045346:Cytochrome b6f Complex; D014357:Trypsin