Reconstitution of Light-independent Protochlorophyllide Reductase from Purified Bchl and BchN-BchB Subunits

Fujita, Yuichi; Bauer, Carl E.

doi:10.1074/jbc.m002904200

Cited by 165 publications

(75 citation statements)

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“…Protochlorophyllide oxidoreductase (DPOR) of R. capsulatus, which reduces ring D of protochlorophyl- lide to form Chlide a, was characterized in vitro with the purified proteins of BchL, BchN, and BchB, supporting a nitrogenase model of the enzyme complex (63)(64)(65). Comparing the primary structures of BchXYZ of COR with BchLNB of DPOR and NifHDK of nitrogenase, significant (ϳ50%) similarities were found to exist between the deduced amino acid sequences of BchX, BchL, and NifH.…”

Section: Discussionmentioning

confidence: 99%

“…Thus, the proteins in common appear to act as ATP-dependent reductase, transferring electrons from suitable sources, including NADH or ferredoxin, to the catalytic complexes composed of the other two subunits. The comparison of the primary structures between BchY-BchZ, BchN-BchB, NifD-NifK, and NifE-NifN reveals less similarity (15,(63)(64)(65), which may reflect their own differing catalytic activities. Our results clearly indicate that BchX and BchY harbor iron-sulfur clusters, but BchZ does not even though it is co-purified with BchY.…”

Section: Discussionmentioning

confidence: 99%

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Superoxide Generation by Chlorophyllide a Reductase of Rhodobacter sphaeroides

Kim

Lee

et al. 2008

Journal of Biological Chemistry

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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...

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Superoxide Generation by Chlorophyllide a Reductase of Rhodobacter sphaeroides

Kim

Lee

et al. 2008

Journal of Biological Chemistry

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“…To quantify pigments UV-visible spectra were measured in 80% acetone employing extinction coefficients of ⑀ 626 ϭ 30.4 mM Ϫ1 cm Ϫ1 for Pchlide (26) and ⑀ 665 ϭ 74.9 mM Ϫ1 cm Ϫ1 for Chlide (27). Circular Dichroism Spectra of ChlL-CD spectra of the purified DPOR subcomplex ChlL 2 at a concentration of 5.5 mg ml Ϫ1 (84 M) in low salt lysis buffer (10 mM HEPES/NaOH, pH 7.5, 15 mM NaCl, and 10 mM MgCl 2 ) were recorded using an anaerobic quartz cuvette (10-mm light path) with a J-810 spectrometer (Jasco) and a thermostated cell holder.…”

Section: Dpor Activity In the Presence Of Atp Analogs-mentioning

confidence: 99%

Biosynthesis of (Bacterio)chlorophylls

Bröcker

Wätzlich

Saggu

et al. 2010

Journal of Biological Chemistry

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Dark operative protochlorophyllide oxidoreductase (DPOR) catalyzes the light-independent two-electron reduction of protochlorophyllide a to form chlorophyllide a, the last common precursor of chlorophyll a and bacteriochlorophyll a biosynthesis. During ATP-dependent DPOR catalysis the homodimeric ChlL 2 subunit carrying a [4Fe-4S] cluster transfers electrons to the corresponding heterotetrameric catalytic subunit (ChlN/ChlB) 2 , which also possesses a redox active [4Fe-4S] cluster. To investigate the transient interaction of both subcomplexes and the resulting electron transfer reactions, the ternary DPOR enzyme holocomplex comprising subunits ChlN, ChlB, and ChlL from the cyanobacterium Prochlorococcus marinus was trapped as an octameric (ChlN/ChlB) 2 (ChlL 2 ) 2 complex after incubation with the nonhydrolyzable ATP analogs adenosine 5-(␥-thio)triphosphate, adenosine 5-(␤,␥-imido)triphosphate, or MgADP in combination with AlF 4 ؊ . Additionally, a mutant ChlL 2 protein, with a deleted Leu 153 in the switch II region also allowed for the formation of a stable octameric complex. Furthermore, efficient complex formation required the presence of protochlorophyllide. Electron paramagnetic resonance spectroscopy of ternary DPOR complexes revealed a reduced [4Fe-4S] cluster located on ChlL 2 , indicating that complete ATP hydrolysis is a prerequisite for intersubunit electron transfer. Circular dichroism spectroscopic experiments indicated nucleotide-dependent conformational changes for ChlL 2 after ATP binding. A nucleotide-dependent switch mechanism triggering ternary complex formation and electron transfer was concluded. From these results a detailed redox cycle for DPOR catalysis was deduced. Reduction of protochlorophyllide a (Pchlide)2 to chlorophyllide a (Chlide) is a central step in the biosynthesis of chlorophyll and bacteriochlorophyll (1). Two evolutionarily unrelated enzymes are capable of catalyzing the stereospecific two-electron reduction of the C17-C18 double bond of Pchlide (Fig. 1A) (2-4). Monomeric, light-dependent Pchlide oxidoreductase (POR; NADPH Pchlide oxidoreductase, EC 1.3.1.33) drives the NADPH-dependent reduction (5-7) of Pchlide bound in the active site via absorption of light energy. This light dependence of POR catalysis prevents angiosperms from synthesizing chlorophyll in the dark (8, 9). Anoxygenicphotosynthetic bacteria make use of a different ATP-dependent Pchlide reducing system, which is termed the light-independent, dark operative Pchlide oxidoreductase (DPOR), whereas gymnosperms, mosses, ferns, algae, and cyanobacteria utilize both POR and DPOR (3). In chlorophyll-synthesizing organisms DPOR is encoded by the chlN, chlB, and chlL genes (10 -12). The corresponding genes for bacteriochlorophyllsynthesizing organisms have been termed bchN, bchB, and bchL (10, 13). DPOR subunits ChlN, ChlB, and ChlL share significant amino acid sequence homologies with nitrogenase subunits NifD, NifK, and NifH, respectively (12,14).In

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“…However, the [4Fe-4S] clusters of (BchNB) 2 have no direct equivalent in nitrogenase. Instead, nitrogenase contains a complicated [8Fe-7S]-P cluster, which interestingly shares all of the four cysteine ligands that have been identified for C. tepidum DPOR (13,16). The two additional cysteine residues involved in P cluster coordination have no counterpart in the sequence of BchNB proteins.…”

mentioning

confidence: 99%

“…The hydrolysis of the two ATP molecules might result in a decrease in the redox potential of the BchL 2 -bound [4Fe-4S] cluster allowing for the transfer of one electron onto a second protein complex composed of BchN and BchB. For this complex a heterotetrameric structure was determined and denoted as (BchNB) 2 (13,15,16). Site-directed mutagenesis experiments of BchN and BchB in combination with kinetic measurements revealed the presence of four cysteine residues crucial for DPOR catalysis.…”

mentioning

confidence: 99%