Superoxide Generation by Chlorophyllide a Reductase of Rhodobacter sphaeroides

Abstract

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.Rhodobacter sphaeroides, a facultative photosynthetic bacterium, contains two SODs 3 ; CuZn-SOD is detected only under the conditions where photosynthetic complexes are formed (1), whereas Fe-SOD is constitutively expressed, although its activity of the aerobically grown cell nearly doubled as compared with that of the anaerobically grown cell. The role of CuZn-SOD in protecting the photoheterotrophic cells from periplasmic superoxide upon exposure to O 2 was proposed (1). A cambialistic SOD using manganese or iron is the only SOD found in Rhodobacter capsulatus and is essential for cell viability (2, 3). Oxidative stress defense in R. sphaeroides and R. capsulatus is also mediated by catalases as well as by glutathione-glutaredoxin and thioredoxin systems that act as thiol-disulfide redox buffer to reduce the protein thiols that were oxidized (4, 5). Mutations in glutathione-glutaredoxin and thioredoxin systems lowered the formation of the photosynthetic complex (4, 5).The formation of photosynthetic complexes of R. sphaeroides is redox-dependent. Lowering oxygen tension induces the formation of intracytoplasmic membrane housing the photosynthetic complexes of R. sphaeroides. Light captured by B800 -850 and B875 LH complexes is transferred to reaction center complex, where the redox reactions are initiated to convert light energy into ATP and reducing power. The oxygenregulated expression of apoproteins of LH and reaction center complexes, which are encoded by puc, puf, and puh operons, was elucidated (for reviews see Refs. 6 and 7).The expression of several enzymes for Bchl a synthesis is also subject to anaerobic induction (8 -10). Protoporphyrin IX, which is synthesized from 5-aminolevulinic acid, is a common in...

Iron-Sulfur Cluster-dependent Catalysis of Chlorophyllide a Oxidoreductase from Roseobacter denitrificans

“…A rhombic S ϭ 1/2 signal was obtained with g values of g 1 ϭ 2.047, g 2 ϭ 1.937, and g 3 ϭ 1.905 located in a region characteristic for [4Fe-4S] 1ϩ clusters (36,37). The observed data are in agreement with related EPR experiments for the L 2 protein of DPOR (4, 38) for (NifH) 2 of Azotobacter vinelandii and also for (BchX) 2 from R. sphaeroides (g 1 ϭ 2.02, g 2 ϭ 1.93 and g 3 ϭ 1.92) (18,39). Similar to what was reported for (NifH) 2 of A. vinelandii (39), no evidence for spin states larger than S ϭ 1/2 (e.g.…”

“…To some extent these theoretical findings are supported by a recent Rhodobacter sphaeroides study where a weak EPR signal was tentatively ascribed to a single 1ϩ cluster on (BchX) 2 or alternatively to 1ϩ clusters located on each BchX protomer (18). The individual BchY protein and also the co-purified (BchY/BchZ) 2 protein complex from R. sphaeroides showed a weak EPR signal indicative for a [3Fe-4S] 1ϩ cluster (18).…”

“…The individual BchY protein and also the co-purified (BchY/BchZ) 2 protein complex from R. sphaeroides showed a weak EPR signal indicative for a [3Fe-4S] 1ϩ cluster (18). However, this cluster type is often found under non-physiological conditions, and a more detailed interpretation would also require the careful analysis of the oligomeric subunit structure of the respective proteins.…”

Competitive Inhibitions of the Chlorophyll Synthase of Synechocystis sp. Strain PCC 6803 by Bacteriochlorophyllide a and the Bacteriochlorophyll Synthase of Rhodobacter sphaeroides by Chlorophyllide a

“…The single-crossover exconjugant (Km r ) was isolated and subjected to segregation for double crossover (bchFNB mutant, Km s ) on Sis medium containing sucrose (15%). In order to disrupt bchZ, the recombinant suicide plasmid pSUPZ100 (16) (26).…”

“…ChlG and ChlP have been thought to emerge through the gene duplication of BchG and BchP, respectively. Recently, we found that the COR reaction, which is specific to Bchl a biosynthesis, generates superoxide at low levels of oxygen (16), and we further proposed that the degeneration of the superoxide-generating COR step may be associated with the emergence of cyanobacterium-based Chl a biosynthesis (15).…”