Ferredoxin-dependent Phenylpyruvate Synthesis by Cell-free Preparations of Photosynthetic Bacteria*

SUMMARY A reductive synthesis of phenylpyruvate from phenyl-acetyl coenzyme A and bicarbonate was obtained in cell-free extracts from the photosynthetic bacterium Chromatium. This phenylpyruvate synthase activity-which appears to be distinct from pyruvate synthase-depends on the reducing power of ferredoxin and requires thiamine pyrophosphate as a coenzyme. Of several ferredoxins assayed, the native ferredoxin from Chromatium was found to be the most effective for phenylpyruvate synthesis. The enzyme extract also catalyzed the conversion of phenylpyruvate to phenylalanine (in the presence of an amino group donor) and the activation of phenylacetate to the phenylacetyl-CoA derivative. Thus, a net synthesis of phenylalanine from phenylacetate and CO2 was accomplished. Closely associated with the phenylpyruvate synthase reaction was an exchange reaction between 14C-bicarbonate and phenylpyruvate. The exchange reaction required thiamine pyrophosphate and CoA as cofactors. Apart from Chromatium, phenylpyruvate and phenylpyruvate-bicarbonate were also found in cell-free extracts from the green photosynthetic bacteria


This phenylpyruvate
synthase activity-which appears to be distinct from pyruvate synthase-depends on the reducing power of ferredoxin and requires thiamine pyrophosphate as a coenzyme.
Of several ferredoxins assayed, the native ferredoxin from Chromatium was found to be the most effective for phenylpyruvate synthesis. The enzyme extract also catalyzed the conversion of phenylpyruvate to phenylalanine (in the presence of an amino group donor) and the activation of phenylacetate to the phenylacetyl-CoA derivative. Thus, a net synthesis of phenylalanine from phenylacetate and CO2 was accomplished. Closely associated with the phenylpyruvate synthase reaction was an exchange reaction between 14C-bicarbonate and phenylpyruvate.
The exchange reaction required thiamine pyrophosphate and CoA as cofactors. Apart from Chromatium, phenylpyruvate synthase and phenylpyruvate-bicarbonate exchange activities were also found in cell-free extracts from the green photosynthetic bacteria Chlorobium thiosulfatophilum and Chloropseudomonas ethylicum.
Previous work in tllis laboratory has revealed a direct l)articil)ation oi" reduced fcrrcdosin in CO2 fixation reactions in bacterial l~l~otosyr~thesis. These rc~action~, driven by the strongly rcducing l)ot,ential of ferrcdosill-about equal to that of molecular hydrogen (1, 2)-involve a reductive carbosylation of an acyl coellzyme :1 derivative to fo1~1 the corresponding cr-keto acid (Aqualion I).
Bachofen, and Arnon in Chromatium (3). Pyruvate s)-ntliaae is now known to be present, in various types of phot,osynthetic (4-G) and nonphotosynthetic anaerobic bacteria ('i-12). h second reaction of this type is the reductive carboxylation of succinyl-CoA to form a-ketoglutarate, which has been found in certain photosynthetic bacteria (4, 6, 13) and has also been 01). served recently in an anaerobic rumen bacterium (14). Another fcrredoxin-dependent reductive carbosylation reaction is the synthesis of oc-ketohutyrate from COY and propionyl-CoR discovered recently by Iluchanan (15). l'yruvate synthase and a-ketoglutarate synthase arc key enzymes in the reductive carboxylic acid cycle (16, 17) , a new cyclic pat.liway for CO* fis:Ltion in bacterial photosynthesis. 21 ~o~~n~~on feature of the three fcrrcdoxin-dependent reductive curboxylation reactions is that the a-keto acids formed are rclatcd to the biosynthesis of amino acids which, in photosynthetic bacteria, constitute the tllain soluble products of COs fixation (18)(19)(20)(21)(22).

METHOlE
Chrornatiun~, strain I), was grown in 13-liter Pyres bottles either in the bicarbon:tte-rn:rlate medium ol Arnon, I&, and Anderson (21) or in the same medium modified by replacing malat e with sodium thiosulfate (final concentration 0.012 11). C. thiosulfatophilum, strnill Tassajara, was grown as described by Evans and Buchanan (5) in :I medium free of acetate. C. et/+ CWL, kindly supplied by Dr. J. M. Olson of the Brookhavcn National Laboratory, was cultured as described bp Evans (6). The harvested cells were stored at -20" prior to use.
The enzyme preparation was freshly made (at 4" under argon or osygen-free ST) for each experiment from 10 g of cell llaste Iesue of July 25, 1971 U. Gehriny and D. I. krnon that, was thawed, suspended ill 10 ml of 0.02 AI IIEPIW buffer, pI-I 7.7 (cont.aining 100 pmoles of dit,hiothreitol), and disrupted by sonication for 2 min with a Ih~nson sonifier (power setting 6). Buffer solutions were rendered anaerobic by bubbling argon gas.
The sonified cell suspension W:M caentrifuged for 10 min at 30,000 x g and the precipitat,e ws discarded.
Ferredosin was removed from the supernatant fluid by passing it t,hrough a DEAE-cellulose cohm~n (2 x 3 cm) c>quilibrated with 0.02 M IIEPES buffer, pII 7.7. -111 effluent volume about equal to the volume applied was collected and centrifuged for 2 hours at about 100,000 x g, and the l"ccil)itate was discarded.
To remove possible interfering subst,rates, the supernatant fluid was passed through a Sephades G-25 column (2 x 36 cm) equilibrated with 0.02 M IIEPES buffer, pII 7.7. The excluded protein fraction was used as the enzyme 1)rcparation.
The protein content was estimated by a modified Biuret method (25) in which a 7 : 2 mixture of acetoize-nlethanol was used instead of trichloroacetic acid for the precipitation of protein. The ferredorins used were isolated in pure form from Clostridium pasteurianum (26)) Chromatium (27)) C. thiosulfatophilum (28), and spinach leaves (1). Ferrcdosin from Azotobacter vinelandii (29) was a gift from I)r. I>. C'. Yoch of this laboratory. Protein concentrations of forredosin solutions were measurctl 1)) the Folin procedure (30) wit,h crysit~lline bovine serum albumin as standard.
Labeled phenylalanine was identified and separated from phenylpyruvate by the thin layer chromatography method (slightly modified) described by Buchanan (15). The identity of the labeled compound with suthcntic phenylalanine was cstablished by the coincidence of the radioactive spot with the spot of authentic phenylalanine as stained with ninhydrin. For the quantitative estimation of phenylnlnninc the radioactive areas of duplicate chromatograms were scral)ctl from the plates, and the material was mixed with scintillation fluid (37) and counted in a scintillat,ion counter.
When phenylacetyl-CoA was generated in the reaction mixture, the rate of phenylpyruvate synthesis was about the same, whether all of the ATP was added initially or whether it was being generated by the creatine phosphate-phosphokinase couple (Treatments 3 and 5, Table TI). In contrast to the pyruvnte synthase (3) and oc-ketobutyrate synthase (15) systems, the addition of semicarbazide as a car-bony1 trapping agent did not increase the synthesis of phenylpyruvate.
The formation of phenylp~ruvate, as measured under the coildit,ions described in Table I, proceeded linearly for about 70 min  and Ivas proportional to the amount of enzyme preparation added  Table  I and for the exchange reaction were as given for the complete treatment in Table VI. up to about 12 mg of protein per 3 ml of reaction mixture. The optimum pII range for phenylpyruvate synthesis was 8.1 to 8.6 (Fig. l), whereas the optimum for the phenylpyruvate-bicarbonate cschange, which is discussed below, was ~1% 7.2.
A comparison of the relative effectiveness of different ferre-  doxins in the phenylpyruvate synthase reaction showed that the ferredoxins from the two photosynthetic bacteria Chromati~m and C. thiosuZjutophiZum were the most effective (Table III). Clostridium ferredoxin was considerably less active, and the effectiveness of the recently isolated new type of ferredoxin from A. &elan&i was intermediate between that of Chromatium and Clostridium.
Spinach ferredoxin, known to be a poor substitute for bacterial ferredosins in the phosphoroclastic cleavage of pyruvate and in the nitrogenase system of C. pasteurianum, wus also virtually without effect in the phenylpyruvate synthase reaction.
The concentration effect of ferredoxin is shown in li'ig. 2. About 180 pg (per 3 ml) of Chromatium ferredoxin saturated the phenylpyruvate syuthase system; Clostridium ferredoxin failed to saturate, even at a concentration of 400 pg per 3 ml. A Lincweaver-Burk plot of the data showed the same Jnlax for both ferredoxins and gave an apparent Km of 5 x 1OP M for Chromafium ferredoxin and 8 x 1OP M for Clostridium ferredosin.
The experiments described so far were carried out with enzyme preparations from Chromatium cells grown autotrophically in a bicarbonate-thiosulfate medium. Table IV shows that comparable levels of phenylpyruvate synthase activity were also present in extracts of cells grown in a malate medium-an observatioc Issue of July 25, 1971 U. Gehring and D. I. dmon t.hat is compatible with the report of Allison and Robinson (23) of equal uptake of 1%~phenylacetate by Chromnti7~m cells grown in either a bicarbonate-thiosulfate or a malate medium. Phenylpyruvate synthase appears to be a constitutive rather than an inducible enzyme of Chromatium cells. Its format:ion was not affected by the addition of phcnylacetate (1 mu) to the culture medium.
S!/nf/~esis of Phenylalanine jrom Phenylacefate--Tlie addition of glutamate or glutamine to the reaction mixture did not alter substantially the total Y-bicarbonate fised but changed drastically the final products formed from pheaylacetnte (Table V).
-\ sharp decrease in 14C-pllen311pyruvatc was accompanied by a marked increase in 14C-pheaylalaaine.
These results show a net cell-free synthesis of phenylalanine from phenylacetate and COZ.
In the presence of either glutamate or glutamine there was also some incorporation of 14C-bicarbonate that could not be accounted for by the methods used.
l'henylpyruvate-Bicarbonate Exchange-Enzyme preparations from Chromatium cells were found to catalyze a 14C exchange reaction between '%-bicarbonate and phenylpyruvate. Incorporation of *4C into phenylpyruvate occurred only when thiamine pyrophosphate and catalytic amounts of CoA were present in the reaction mixture (Table VI).
Coh could not be replaced by any of the other sulfhydryl compounds tested. However, a vigorous exchange occurred without added Co.4 when the system was illuminated in the presence of chloroplasts, i.e. under conditions that could have brought about the photoreduction of any ferredosin present,. Although no ferredosin was added in this case, it seems likely that the &ivation of the eschangc reaction by illumination was due t,o the reduction of trace amounts of ferredosin that remained in the enzyme preparation after it was passed through the DESE-cellulose column.
The possibility that reduced ferredosin did indeed replace CoA was strengt'hencd by the observed stimulation of the exchange reaction upon adding ferredosin to the illuminated reaction mixture. aidded NADFI or N;XJ)PI-I was wholly ineffective as a substitute for CoA but some exchange activity occurred in the presence of sodium dithionite, possibly due to the reduction of traces of ferredosin that remained in the reaction mixture.
The pH curve for t.he COB-catalyzed exchange react.ion had a relatively flat drop on the acid side of the optimum pH of 7.2 ( Fig. 1)-a distinctly different pH profile from that of the phenyl-pyru\ratc synthase reaction.
At their rcspectire pl I optima, t.he rate of the exchange react,ion was about three times greater than the rate of phenylpyruvate synthesis.
Phenylpyruvate Synthase T'ersus P~r7~vafe Synthnse-I'hcnylpyruvate s)-nthase appears to be distinct from pyruvate synthase. This view is supported by (a) the presence of the pyruvate but not the phenylpgruvate enzyme in C. pasteurianum (see below), (b) differences in stability of phenylpyruvate synthase and pyruvate synthase in Chromatium cells stored at -20" (after 4 months of storage, some cells had no phenylpyruvate synthase activity while retaining a high level of pyruvate synthase activity), and (c) differential requirements of thiamine pyrophosphatc.
Phenylpyruvate synthesis by cell-free extracts of Chromatium had a distinct requirement for thiamine pyrophosphate (Table I). By contrast, pyruvate synt,hnse of Chromatium shows such a requirement. only after the endogenous thiamine pyrophosphate is removed by special treatment,s (38).
The replacements of CoA incllldsd 10 pmoles of dithiothrcitol or 20 Hmoles each of one of the compounds listed.  Table I). These cell-free extracts also showed phenylpyruvate-bicarbonate exchange activity. Thus, the phenylpyruvate-bicarbonate exchange activity appears to be RIways closely associated with phenylpyruvate synthase. Neither phenylpyruvate synthase nor phenylpyruvate-bicarbonate eschange activity was detected in cell-free extracts of the nonphotosynthetic anaerobe C. pasfeurianum-an organism that contains an active pyruvate synthase system (7).