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 degrees C under aerobic conditions and decreased by 10-15% when they were stored for 6 days at 4 degrees C 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 micromol NADP(+) micromol BChl a(-1) h(-1)): FdA, 2.0 microm and 258; FdB, 0.49 microM and 304; FdC, 1.13 microM and 226; FdD, 0.5 microM and 242; spinach Fd, 0.54 microM and 183. The V(max) value of FdB was more than twice that previously reported for purified RC preparations from green sulfur bacteria.
Seo
D
D
Department of Biology, School of Education, Waseda University, Tokyo, Japan. 699L5097@mn.waseda.ac.jp
Tomioka
A
A
Kusumoto
N
N
Kamo
M
M
Enami
I
I
Sakurai
H
H
D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D019414:Chlorobi / Q000737:chemistry*; D002852:Chromatography, Ion Exchange; D005288:Ferredoxins / Q000737:chemistry / Q000302:isolation & purification*; D007700:Kinetics; D008969:Molecular Sequence Data; D009249:NADP / Q000737:chemistry; D010084:Oxidation-Reduction; D010788:Photosynthesis; D045322:Photosynthetic Reaction Center Complex Proteins / Q000737:chemistry / Q000302:isolation & purification*; D045331:Photosystem I Protein Complex; D013053:Spectrophotometry
D001426:Bacterial Proteins; D005288:Ferredoxins; D045322:Photosynthetic Reaction Center Complex Proteins; D045331:Photosystem I Protein Complex; C096017:PscA protein, green sulfur bacteria; D009249:NADP