/. Biochem., 79, 361-371 (1976)
Green Photosynthetic Bacterium Prosthecochloris aestuarii Yuzo SHIOI,1 Ken-ichiro TAKAMIYA, and Mitsuo NISHIMURA Department of Biology, Faculty of Science, Kyushu University, Higashi-ku, Fukuoka, Fukuoka 812 Received for publication, August 25, 1975
NAD+ reductase of the green photosynthetic bacterium Prosthecochloris aestuarii was isolated and purified by ammonium sulfate fractionation, DEAE-cellulose column chromatography, and Sephadex G-200 gel filtration. This enzyme is an FAD-containing flavoprotein and has absorption maxima at 485 (shoulder), 452, 411, and 385 nm (the 411 nm band is due to cytochrome). The molecular weight of the enzyme as determined by gel filtration using Sephadex G-200 is 119,000. The enzyme catalyzes the reduction of NAD+ and NADP+ by photoreduced spinach ferredoxin or reduced benzyl viologen. It also catalyzes the reduction of cytochromes and dyes such as benzyl viologen and 2, 6-dichloroindophenol (DCIP) with NADH or NADPH as the electron donor. The reduction of cytochrome c-555(550) of this organism is accelerated by the addition of cofactors such as menadione. In these reactions, the enzyme is more specific for NAD+ or NADH than for NADP+ or NADPH. Reduction of cytochrome c or dyes catalyzed by the enzyme is strongly inhibited by rotenone or amytal, but not by antimycin A or o-phenanthroline. It is suggested that NAD+ photoreduction in this organism takes place via direct electron flow with the mediation of ferredoxin and flavoprotein enzyme, as in Chlorobium and green plants.
The mechanism of pyridine nucleotide reduction in green plants has been intensively studied by various investigators (1—5). It is known that NADP+ is photochemically reduced in green plants by direct electron transfer from photosystem I via ferredoxin and ferredoxinNADP+ reductase [EC 1.6.7.1]. The latter enzyme is an FAD-containing flavoprotein which also catalyzes the reduction of cyto1
Present address: Department of Biology, Medical College of Miyazaki, Miyazaki 889-16. Abbreviation: DCIP, 2,6-dichloroindophenol. Vol. 79, No. 2, 1976
361
chrome/, NAD+ and dyes such as ferricyanide, DCIP, and benzyl viologen by NADPH. The same mechanism of NADP+ reduction operates in various algae such as blue green alga (6), diatom (7), and green alga ( 1.5x100 cm) at 20°. purified and characterized from purple bacteria Bovine pancreas chymotrypsinogen (molecular Rhodopseudomonas palustris (20). weight 22,000), bovine serum albumin (molecThis paper deals with the purification and ular weight 65,000), and ^-globulin (molecular characterization of pyridine nucleotide reduc- weight 160,000) were used as markers. The tase from a green photosynthetic bacterium, moving phase consisted of a solution of 0.05 M Prosthecochloris aestuarii. The enzyme showed Tris-HCl buffer (pH 7.5) containing 0.1 M KC1. various properties similar to those of the The void volume was estimated by using Blue Chlorobium enzyme. However, unlike the Dextran 2000. Protein in each fraction was measuring the absorbance at 280 Chlorobium enzyme, this enzyme was more monitored by + + + nm. NAD reductase was followed in terms specific for NAD or NADH than for NADP or NADPH and reduction of cytochrome c-555 of NADH-DCIP reductase activity (see Assay (550) of this organism with NADH was ac- of Enzymatic Activities). celerated by the addition of a cofactor such as Isoelectric Point Determination—The isomenadione. electric point was determined by the isoelectric focusing procedure described by Vesterberg and Svensson (27), using an Ampholine colMATERIALS AND METHODS umn and carrier Ampholite (pH range 3.5—10, Organism and Growth Conditions — The LKB-Produkter AB, Sweden) at 4° for 48 hr. green photosynthetic bacterium Prosthecochloris After isoelectric focusing, the reductase was aestuarii was reisolated and identified by Pro- determined in terms of NADH-DCIP reductase fessor N. Pfennig of the University of Got- activity in each 2 ml fraction (see Assay of tingen from the culture previously identified Enzymatic Activities). pH measurement was as Chloropseudomonas ethylica strain 2-K.1 performed at 2° with a Hitachi-Horiba pH meter F-5SS equipped with a temperature compensator. 1 The name of the species used in our previous Determination of Flavin—The prosthetic reports (21, 22) should therefore be corrected to ProstheaxMoris aestuarii from Chloropseudomonas group of the reductase, flavin, was determined by the method of DeLuca et al. (28). The ethylica strain 2-K. linked nature was studied in detail by Keister and Yike (10) and several other investigators (77, 12). This reaction was also found in several other species of Rhodospirillaceae (13—
NAD* REDUCTASE OF Prosthtcochloris aestuarii
Preparation of Cytochromes — Prosthecochloris cytochromes c-551.5 and c-555(550) were prepared by the method described in our previous paper (21). Euglena cytochrome c-552 was kindly supplied by Dr. K. Wakamatsu (Fukuoka Women's Univ., Fukuoka). Determination of Protein — Protein was determined by the method of Lowry et al. (32), using bovine serum albumin as a standard. Reagents—FAD, FMN, AMP, D-amino acid oxidase, catalase [EC 1.11.1.6], bovine pancreas chymotrypsinogen, bovine serum albumin, 7-globulin, and rotenone were purchased from Sigma Chemical Co. (U.S.A.). NAD+, Vol. 79, No. 2, 1976
NADP+, NADH, NADPH, lactate dehydrogenase [EC 1.1.1.27], horse heart cytochrome c, and DL-alanine were purchased from Boehringer Mannheim Co. (West Germany); DEAEcellulose (0.3 meq/g) was from Brown Co. (U.S.A.); Sephadex G-200 and Blue Dextran 2000 were from Pharmacia Fine Chemicals (Sweden). All other reagents were of analytical grade. RESULTS
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activation of the apo-enzyme of D-amino acid oxidase [EC 1.4.3.3] by flavin solution extracted from the NAD+ reductase was measured. The apo-enzyme of D-amino acid oxidase, prepared by repeated dialysis against ammonium sulfate solution, is known to be activated only by FAD (28). Flavin solution used for the assay was prepared by treating the enzyme with trichloroacetic acid by the method of Kusai and Yamanaka (19). Assay of Enzymatic Activities—NAD+ reductase activity was determined as described by Yamanaka and Kamen (7) with photoreduced spinach ferredoxin or dithionite-reduced benzyl viologen as the electron donor. Spinach chloroplast fragments partially lacking in ferredoxin and NADP+ reductase were prepared by the method of Shin (29) under milder conditions (2—3 hr at 4°). Purified spinach ferredoxin was prepared by the method of Tagawa and Arnon (30). The chlorophyll content of chloroplast fragments was determined in 80% acetone extract using the absorption coefficient of Mackinney (31). NADH(NADPH)-cytochrome c reductase activity was measured anaerobically (under argon) or aerobically at 20° by following the increase in absorbance at the a-band of the cytochrome. NADH(NADPH>benzyl viologen reductase activity was determined anaerobically (under argon) at 20° by measuring the increase in absorbance at 550 nm. NADH(NADPH)-DCIP reductase activity was measured aerobically at 20° by following the decrease in absorbance at 600 nm.
363
Isolation and Purification of NAD* Reductase — Frozen cell paste (120—150 g wet weight) was thawed and suspended in 500 ml of 0.05 M Tris-HCl buffer, pH 7.8. The cells were disrupted by passing them through a French pressure cell (Ootake Co.) twice at 1,500 kg/cm2 at 0—5°. The suspension of broken cells was centrifuged at 10,000 xg for 20 min to remove cell debris and intact cells. The green supernatant solution was further centrifuged at 144,000X0 for 90 min. The brownish red supernatant solution obtained was fractionated with ammonium sulfate between 40 and 80% saturation. The precipitate was collected by centrifugation and dissolved in a small volume of 0.05M Tris-HCl buffer, pH 7.8. The resulting solution was dialyzed against 3 liters of 0.05 M Tris-HCl buffer, pH 7.8, containing 0.1 M NaCl. The dialyzed solution was loaded onto a DEAE-cellulose column ( 5x10 cm) previously equilibrated with the same buffer. The column was washed with the same buffer to remove unadsorbed proteins. Then, the enzyme was eluted with 0.1 M Tris-HCl buffer, pH 7.8, containing a linear gradient of NaCl from 0.1 M to 0.4 M. The enzyme was eluted at a concentration of 0.23-0.26 M. The eluate was fractionated with ammonium sulfate between 45 and 65% saturation. The precipitate was collected by centrifugation and dissolved in a small volume of 0.05 M Tris-HCl buffer, pH 7.8. The resulting solution was further purified by molecular sieving with Sephadex G-200 ( 3.5x70 cm). The enzyme was developed again on the Sephadex G-200 column and the main fraction obtained was used as the enzyme preparation. Elution patterns of the reductase activity and
364
Y. SHIOI, K. TAKAMIYA, and M. NISHIMURA
TABLE I. Distribution of NAD+ reductase. Fresh cells (2-day culture) were suspended in 0.05 M TrisHC1 buffer, pH 7.5, and disrupted by passing once through a French pressure cell at 1,500 kg/cm*. Debris and intact cells were precipitated by centrifugation at 10,000 XQ for 20 min. The resulting supernatant solution was further centrifuged at 144,000X(; for 60 min to separate soluble and particulate fractions. NADH-benzyl viologen reductase activity was measured as described in " METHODS." The reaction mixture was as follows; 50 //moles of Tris-HCl buffer, pH 7.5, 0.2 //mole of benzyl viologen, 0.1 fimole of NADH, an appropriate amount (0.1 ml) of enzyme fraction and water to make a total volume of 2.5 ml. Fractions
spinach, which was released from the chloroplast membranes by continuous stirring (29). These data suggest that the Prosthecochloris NAD+ reductase is weakly bound to the membrane structure, like that of Chlorobium or green plants. Spectral Properties—Absorption spectra of the oxidized and reduced forms of purified NAD+ reductase are shown in Fig. 1. The spectra had absorption maxima at 452, 411, and 385 nm and a shoulder around 485 nm in the oxidized state and a maximum at 419 nm in the reduced state. These bands suggest the flavoprotein nature of the enzyme. However, absorption bands at 411 nm and 419 nm may be attributed to contaminating cytochrome in the preparation (Soret band of the oxidized and reduced forms, respectively). The enzyme was isolated in an almost completely oxidized state and the reduced enzyme was oxidized by aeration. Prosthetic Group—The trichloroacetic acid extract of the NAD+ reductase was yellow in color. Addition of the extract to the apoenzyme of D-amino acid oxidase resulted in recovery of the enzyme activity lost on removal of the prosthetic group, FAD. Addition of authentic FAD also effectively restored the activity, but FMN did not (Fig. 2). Furthermore, reactivation of the NADH-benzyl viologen reductase activity of the trichloroacetic acid-treated Prosthecochloris reductase was studied by the method of Kusai and Yamanaka
Total activity Specific activity /nmoles/minA (/imoles/min) \ m g protein/
Homogenate Debris and intact cells Particulate Soluble
7.74 0.83 3.88 3.48
19.8 11.4 28.4 18.9
400
500 WAVELENGTH ( n m )
Fig. 1. Absorption spectra of NAD+ reductase. The enzyme was dissolved in 0.05 M Tris-HCl buffer, pH 7.8 (1 mg protein/ml). , oxidized form; , reduced with / . Biochem.
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the protein absorption (at 280 nm) after isoelectric fractionation in an Ampholine column were practically identical (cf. Fig. 4). No major protein band was observed in the elution diagram except for the reductase band and a trace band due to contaminating cytochrome. Crude and purified NAD+ reductase was stable for several months when stored at —15°. However, the enzyme lost its activity on repeated freezing and thawing. When the enzyme solution was heated for a brief period {e.g. at 55° for 5 min), the enzyme lost most of its activity. Distribution of NAD+ Reductase—The distribution of NAD+ reductase was studied in the homogenate and three fractions obtained after the French press treatment (1,500 kg/cm1) and subsequent centrifugations, using NADHbenzyl viologen reductase activity as an indicator of the enzymatic activity (Table I). After the French press treatment, about 50% of the total activity appeared in the particulate fraction with a similar amount in the soluble fraction. A similar distribution was observed with Chlorobium thiosulfatophilum (18, 33) and with the NADP+ reductase of
NAD+ REDUCTASE OF Prosthecochloris aestuarii
365 3J0
03
Serum albumin
. NAD*-reductase
/•-Globulin
uo
4.0
5
10 Time(min)
Fig. 2. Reactivation of the apo-enzyme of r>amino acid oxidase by flavins. Procedures are described in the text. O : authentic FAD (1 fiM) was added. • • : trichloroacetic acid extract of the NAD+ reductase was added. Trichloroacetic acid was removed by ether extraction. The absorbance of the yellow trichloroacetic acid extract at 450 nm was about 0.003, corresponding to a concentration of about 0.3 fiM assuming that the light-absorbing component in the extract was FAD. D : authentic FMN (1 fiu) was added.
10
20
45
5.0 5.5 log Molecular weight
Fig. 3. Plot of the ratio of elution volume (K«) to void volume (Ko) against the logarithm of the molecular weights for marker proteins and Prosthecochloris NAD+ reductase on Sephadex G-200. The column (fi 1.5x100 cm) was equilibrated with 0.05 M Tris-HCl buffer, pH 7.5, containing 0.1 M KC1. (19). The activity of the apo-enzyme of the reductase was recovered on addition of FAD (1 /*M) or the trichloroacetic acid extract of the reductase. FMN (1 ftM) did not restore the activity (data not shown). From these results, the prosthetic group of the NAD+ reductase was concluded to be FAD. Molecular Weight—A plot of the ratio of the elution volume (V,) to the void volume
30 Fractions
40
50
60
Fig. 4. Elution diagram of Prosthecochloris NAD+ reductase after isoelectric fractionation. • , Reductase monitored in terms of the NADH-DCIP reductase activity in each of the 2 ml fractions (0.2 ml aliquots were used in the enzymatic assay). O, pH measured at 2° in each fraction. Vol.L79, No. 2, 1976
6.0
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iChymotrypsinogen
366
Y. SHIOI, K. TAKAMIYA, and M. NISHIMURA
The NAD+ reductase also reduced both NAD+ and NADP + with reduced benzyl viologen as an electron donor (Table III). The rate of
TABLE II. Light-dependent reduction of pyridine nucleotides catalyzed by NAD+ reductase. The standard reaction mixture consisted of 40 //moles of Tris-HCl buffer, pH 7.8, washed chloroplast fragments equivalent to 30 //g of chlorophyll, 4 nmoles of spinach ferredoxin, 0.4 //mole of NAD+ or NADP*, 80 nmoles of DCIP, 4 //moles of sodium ascorbate, and 622 f/g of the enzyme in a total volume of 2.5 ml. The reaction was carried out anaerobically (under argon) at 20° under illumination of 35,000 lux. Reaction mixture
NAD+ reduced (nmoles/5 min)
Light Complete Ferredoxin omitted Ascorbate-DCIP omitted Chloroplasts omitted NAD+ or NADP+ omitted Enzyme omitted Dark Complete
NADP+ reduced (nmoles/5 min)
69.3
97.0
21.6
—
16.1
—
0.0
—
0.0
0.0
13.7
79.5
0.0
0.0
NAD+ reduction with photoreduced ferredoxin as an electron donor (on an enzyme basis) was considerably slow compared to that with reduced benzyl viologen. The Prosthecochloris enzyme reduced NAD+ more rapidly than NADP+, in contrast to the Chlorobium enzyme (19).
TABLE III. Pyridine nucleotide reduction with reduced benzyl viologen as an electron donor. The standard reaction mixture contained 150 //moles of Tris-HCl buffer, pH 7.8, 1.0 //mole of benzyl viologen, 0.5 //mole of NAD+ or NADP+, and 579 //g of the enzyme in a total volume of 2.5 ml. Benzyl viologen was carefully reduced by adding a few crystals of NajS,O4 (less than one equivalent with respect to viologen) before the reaction was started. Oxidized benzyl viologen and reduced pyridine nucleotide were determined according to Ref. 7. The reactions were carried out anaerobically at 20°. Reaction mixture Complete Enzyme omitted NAD+ omitted NAD+ omitted, NADP+ added
n 0.310 0.049 0.037 0.061
Benzyl viologen oxidized (nmoles/min)
.dAuo nm/min
86.4 13.6 10.3 16.9
0.105 0.016 0.010 0.028
Pyridine nucleotide reduced (nmoles/min) 42.2 6.4 4.0
11.2
J. Biochtm.
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(Vo) against the logarithm of the molecular weights of the marker proteins is shown in Fig. 3. Points in this plot gave a good straight line. A molecular weight of 119,000 was calculated for the reductase from this plot. Isoelectric Point—As shown in Fig. 4, the isoelectric point of Prosthecochloris reductase was determined to be 4.71 at 2°, by the electrofocusing technique with carrier Ampholite covering the pH range 3.5—10. Reduction of NAD+ and NADP+—NAD+ and NADP + were reduced by purified Prosthecochloris NAD+ reductase in the presence of photoreduced spinach ferredoxin as the electron donor (Table II). The net rate of NAD+ reduction (complete minus enzyme-omitted) by the enzyme was 3—4 times that of NADP+, although fluctuations were observed in the case of NADP + reduction. The reduction was dependent on NAD+ reductase and reduced ferredoxin, the latter being formed by illuminating chloroplasts in the presence of a suitable electron-donating system (ascorbate plus DCIP). Chloroplast fragments used in the reaction still retained some NADP+ reductase and ferredoxin, as shown by NADP+ photoreduction in the absence of the Prosthecochloris enzyme. Spinach ferredoxin was used in these experiments, because it is known that ferredoxin isolated from green photosynthetic bacteria is not stable at any level of purity (34).
:NAD+ REDUCTASE OF Prosthtcochloris aestuarii
TABLE IV. NADH(NADPH)-cytochrome c reductase -activity of NAD+ reductase. The activity was measured aerobically at 20° by recording the increase in absorbance at 550 nm. The standard reaction mixture contained 50 //moles of Tris-HCl buffer, pH 7.8, •0.04 //mole of horse heart cytochrome c, 0.1 //mole of NADH or NADPH and 83 //g of enzyme in a total volume of 2.0 ml.
Additions
Concentration (//M)
None Menadione
—
10 20
Benzyl viologen
10 20
PAD
1 10
PMN
1 10
"Riboflavin
1 10
Vol. 79, No. 2, 1976
Cytochrome reduced (^Mwonm i/min) NADH
NADPH
0.044 0.260 0.585
0.012 0.020 0.038
0.116 0.178 0.063 0.118 0.066 0.081 0.060
0.017 0.022 0.016
0.079
0.013
0.015
cochloris cytochrome c-551.5 was not reduced in the presence or absence of menadione. The effects of inhibitors on NADH-cytochrome c reductase activity are shown in Table VI. Flavoprotein inhibitors such as TABLE V. Reduction of cytochromes from various sources with NADH as an electron donor. The reaction mixture and conditions were the same as in Table IV, except for the sources of cytochrome c's. AAajraxa
Source of cytochrome
a-Peak
No addition
Menadione added (10 //M)
550
0.045
0.263
552
0.300
0.303
555
0.009
0.167
551.5
0
0
(nm) 1
Horse heart Euglena* Prosthecochloris aestuariib Prosthtcochloris aestuariib 1
The reaction was carried out aerobically. b The reaction was carried out anaerobically in order to prevent autooxidation. TABLE VI. Effects of inhibitors on NADH-cytochrome c reductase activity. The reaction mixture and other conditions were the same as in Table IV, except for the addition of 10 //M menadione. Inhibitors None Rotenone Atebrin
Concentration (mM) —
100
0.005 0.05
52
0.05 0.1
Amytal Phenylmercuric acetate
0.05 0.05
AMP
2.0
NAD+ NADP+ Antimycin A o-Phenanthroline Carbonylcyanide m-chlorophenylhydrazone
Activity / % of N \controly
46 61 52 52 22 66
0.05
79
0.5
71
0.05 0.05 0.05
98
nV. nni \AJ±
91 109
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NADH-Cytochrome c Reductase Activity— The NAD+ reductase showed NADH(NADPH>cytochrome c reductase activity (Table IV). High NADH-cytochrome c reductase activity was observed in the presence of menadione, l>ut flavins were less effective. As an electron •donor, NADH reduced cytochrome c more rapidly than NADPH. NADPH was almost ineffective even in the presence of menadione or benzyl viologen. The pH optimum for cytochrome c reduction by NADH was 8.9 and Km values for NADH and NADPH were 4.3 ftM. and 260 fiM, respectively (Table VIII). The Prosthecochloris enzyme catalyzed the reduction of cytochrome c's from various sources -with NADH as an electron donor (Table V). Euglena cytochrome c-552 was the most efficient •electron acceptor in the absence of menadione. The reduction rate of Euglena cytochrome c552 wai not affected by the addition of menadione. The reduction of cytochrome c-555(550) •of this organism was slow, but it was accelerated t>y the addition of menadione. Prosthe-
367
Y. SHIOI, K. TAKAMIYA, and M. NISHIMURA
368
Additions
Concentration
None FAD
Benzyl viologen reduced (J/lwonm/min)
(//M)
NADH
NADPH
NADH
NADPH
—
0.420 0.440 0.420 0.425 0.263 0.420
0.036 0.038
0.625 0.660 0.724 0.645 0.740 0.632 0.739
0.027
1 10
FMN
1 10
Riboflavin Menadione
DCIP reduced ( — 4i4»oo nm/min)
1
0.036 0.036
100
rotenone, atebrin, or amytal strongly inhibited the reduction of cytochrome c in the presence of menadione. Half-inhibition concentrations were 0.05 mM for rotenone and amytal and 0.1 mM for atebrin. However, the activity was not affected by antimycin A or o-phenanthroline. In addition to flavoprotein inhibitors, carbonyl cyanide w-chlorophenylhydrazone and phenylmercuric acetate were potent inhibitors. NAD+ and AMP (at high concentrations) also
-i
n
NADH
inhibited cytochrome c reduction. Similar results were obtained with Chlorobium NAD(P)+ reductase by Kusai and Yamanaka (19). NADH(NADPH) - Benzyl Viologen and -DCIP Reductase Activities — The Prosthecochloris reductase catalyzed the reduction of dyes by NADH or NADPH (diaphorase activity) (Table VII). The optimum pH in NADHDCIP reduction was 8.9. Benzyl viologen reduction by NADH was hardly stimulated byflavins but DCIP reduction was slightly stimulated by FAD and FMN. Though it is effective in the reduction of mammalian cytochrome c, menadione was not very effective in DCIP reduction. Dye-diaphorase activities were inhibited by flavoprotein inhibitors, but
c E TABLE VIII. Values of Km for NAD(P)H in the cytochrome c- and dye-reductase activities of NAD+ reductase. Basic reaction mixtures and conditions were the same as in Tables VI and VII, except for NAD(P)H concentrations.
£
Jo.2 i
NADPH
L
0
•-