Purification of ferredoxins and their reaction with purified reaction center complex from the green sulfur bacterium Chlorobium tepidum

Four ferredoxin (Fd) fractions, namely, FdA-D were purified from the green sulfur bacterium Chlorobium tepidum. Their absorption spectra are typical of 2[4Fe-4S] cluster type Fds with peaks at about 385 and 280 nm and a shoulder at about 305 nm. The A(385)/A(280) ratios of the purified Fds were 0.76-0.80. Analysis of the N-terminal amino acid sequences of these Fds (15-25 residues) revealed that those of FdA and FdB completely agree with those deduced from the genes, fdx3 and fdx2, respectively, found in this bacterium (Chung and Bryant, personal communication). The N-terminal amino acid sequences of FdC and FdD (15 residues) were identical, and agree with that deduced from the gene fdx1 (Chung and Bryant, personal communication). The A(385) values of these Fds were unchanged when they were stored for a month at -80 degreesC under aerobic conditions and decreased by 10-15% when they were stored for 6 days at 4 degreesC under aerobic conditions, indicating that they are not extremely unstable. In the presence of Fd-NADP(+) reductase from spinach, and a purified reaction center (RC) preparation from C. tepidum composed of five kinds of polypeptides, these Fds supported the photoreduction of NADP(+) at room temperature with the following K-m and V-max (in mu mol NADP(+) mu mol BChl a(-1) h(-1)): FdA, 2.0 muM and 258; FdB, 0.49 muM and 304; FdC, 1.13 muM and 226; FdD, 0.5 muM and 242; spinach Fd, 0.54 muM and 183. The V-max value of FdB was more than twice that previously reported for purified RC preparations from green sulfur bacteria. (C) 2001 Elsevier Science B.V. All rights reserved.