Superoxide generation by chlorophyllide a reductase of Rhodobacter sphaeroides.
Chlorophyllide a reductase of Rhodobacter sphaeroides, which were reconstituted with the purified subunits of BchX, BchY, and BchZ, reduced ring B of chlorophyllide a using NADH under anaerobic conditions. Interestingly, suppressor mutations rescuing the inability of R. sphaeroides Fe-SOD mutant to grow in succinate-based minimal medium were predominantly mapped to BchZ subunit of chlorophyllide a reductase. The enzyme is labile in the presence of O(2). However, it generates superoxide at low O(2). The enzymes reconstituted with BchX, BchY, and the mutein subunit of BchZ from suppressor mutants showed less activity not only for chlorophyllide a reduction but also for superoxide generation compared with the enzyme reconstituted with the wild-type subunits. BchX, which contains FMN, and BchY are iron-sulfur proteins, whereas BchZ is a hemoprotein containing b-type heme. Neither chlorophyllide a reduction nor superoxide generation was observed with the enzyme reconstituted with the wild-type subunits of BchX and BchY, and the apo-subunit of BchZ that had been refolded without heme, in which FMN of BchX was fully reduced. Thus, superoxide is generated not from FMN of BchX but from heme of BchZ. Consistently, the heme of BchZ muteins was half-reduced in its redox state compared with that of wild-type BchZ.
Kim
Eui-Jin
EJ
Department of Life Science and Interdisciplinary Program of Integrated Biotechnology, Sogang University, Seoul 121-742, Korea.
Kim
Ju-Sim
JS
Lee
Il-Han
IH
Rhee
Hae J
HJ
Lee
Jeong K
JK
D001483:Base Sequence; D003470:Culture Media; D017931:DNA Primers; D004578:Electron Spin Resonance Spectroscopy; D004591:Electrophoresis, Polyacrylamide Gel; D016296:Mutagenesis; D044925:Oxidoreductases Acting on CH-CH Group Donors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010100:Oxygen / Q000378:metabolism; D012242:Rhodobacter sphaeroides / Q000201:enzymology*; D013481:Superoxides / Q000378:metabolism*
D003470:Culture Media; D017931:DNA Primers; D013481:Superoxides; D044925:Oxidoreductases Acting on CH-CH Group Donors; D010100:Oxygen