JOURNAL OF BACTERIOLOGY, Sept. 1968, p. 678-686Copyright © 1968 American Soziety for Microbiology

Vol. 96, No. 3Printed in U.S.A.

Oxidation of D(-) Lactate by the Electron TransportFraction of Azotobacter vinelandii

PETER JURTSHUK AND LAHOMA HARPERDepartment of Microbiology, The University of Texas, Austin, Texas 78712

Received for publication 8 June 1968

D(-) Lactate oxidation in Azobacter vinelandii strain 0 is readily carried out bythe membrane bound enzyme that concentrates in the electron transport fraction(R3). This oxidation in the R3 fraction is not dependent on externally added nico-tinamide adenine dinucleotide, flavine adenine dinucleotide, or flavine mononucleo-tide. Phenazine methosulfate, 02, and menadione all served as good electroncarriers, and the oxidation of lactate was limited to the D(-) stereoisomer. Of all thea-hydroxyacids examined, only D(-) lactate and D(-) a-hydroxybutyrate wereoxidized by the R3 fraction. Paper chromatographic studies revealed that the 2,4-dinitrophenylhydrazine derivative formed from D( -) lactate oxidation was pyruvate.Pyruvate, in turn, could be further decarboxylated nonoxidatively by the R3 frac-tion. Spectral studies revealed that both the R3 flavoprotein and cytochrome (a2,a,, bi, c4, and c5) components were reduced by D(-) lactate. The D(-) lactic oxi-dase activity was sensitive to electron transport inhibitors, i.e., chlorpromazine,antimycin A, 2-n-heptyl-4-hydroxyquinoline-N-oxide, rotenone, dicumarol, andcyanide, and to a small extent to 4,4,4-trifluoro-1-(2-thienyl)-1,3-butane-dione(TFTB) and Amytal. The D(-) lactic phenazine methosulfate and menadionereductases were sensitive only to dicumarol and TFTB. Chlorpromazine wasfound to be a highly specific inhibitor of D(-) lactic oxidase activity, 50% in-hibition occurring at 6.6 X 10-6 M.

The oxidation of lactate has long been observedin both mammalian and microbial systems. Inmammalian tissue, and in some microbial sys-tems, lactate oxidation is primarily nicotinamideadenine dinucleotide-dependent (34) and is spe-cific for the L(+) stereoisomer (19) although theoxidation of D(-) a-hydroxyacids is known tooccur to some extent in animal tissue (36). Inaddition, a flavoprotein-dependent type of lactateoxidation has been observed also which has beenstudied almost exclusively in microorganisms. Theflavoprotein catalyzed reaction can be specific foreither the L(+) or D(-) isomer of lactate. Thistype of flavoprotein dehydrogenase is usuallyassociated with the intracytoplasmic membraneswhich also contain large concentrations of cyto-chromes (20, 26). Those organisms that have beenreported to carry out the flavoprotein-dependentlactate oxidation are: Aerobacter aerogenes (27),Acetobacter peroxydans (5), Azotobacter vinelandii(4, 14, 20, 38), Bacillus megaterium (33), Escher-ichia coli (2, 13), Saccharomyces cerevisiae (1, 3,10, 24, 30, and 31), Lactobacillus arabinosus (32),Leuconostoc mesenteroides (17), Mycobacteriumphlei (6, 35), M. tuberculosis var. hominis (21),M. avium (6, 39), Propionibacterium pentosaceum(23), Pseudomonas natriegens (37), Staphylococcus

aureus (22), and the ciliated protozoan, Tetrahy-mena pyriformis (7). A notable exception is theparticulate fraction isolated from Leuconostocmesenteroides which has been reported to carryout the oxidation of lactate in the absence offlavoprotein and pyridine nucleotide (17).

Preliminary investigations have indicated thatlactate oxidation in A. vinelandii strain 0 is car-ried out by a particulate flavoprotein oxidore-ductase that is associated with the electrontransport fraction (4, 14, 20, 38). The Azotobacterelectron transport fraction catalyzes the oxidationof reduced nicotinamide adenine dinucleotidephosphate, succinate, tetramethyl-p-phenylenedia-mine, and cytochrome c (4, 14, 15, 16, 20, 38) andis in many waysfunctionally similar to the electrontransport system of mammalian mitochrondria.A major difference between the two particulateelectron transport systems is that in Azotobacterspp. there are several other flavoprotein-depend-ent oxidoreductases, which can carry out theoxidation of lactate and malate (4, 14, 20; P.Jurtshuk, C. H. Denton, and A. J. Bednarz, Bac-teriol. Proc., 1967, p. 104), as well as an activehydrogenase (9). The purpose of this investigationwas to examine and characterize the type oflactate oxidation that occurred in cell-free ex-

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tracts of A. vinelandii strain 0. Although it waspreviously shown that lactate oxidation in Azoto-bacter spp. was associated with the particulatefractions of cell-free extracts, the type of enzymeresponsible for this oxidation has not been char-acterized with regard to the type of isomeroxidized, overall reaction that occurred, type ofcofactors and acceptors required, and the effectof various inhibitors.

MATERIALS AND METHODS

Chemicals. The following materials were obtainedfrom the sources indicated: D(-) lactic acid (crystal-line), D,L-a-hydroxyisobutyric acid, D,L-a-hydrox-yisovaleric acid, D,L-a-hydroxycaproic acid, Amytal,antimycin A, dicumarol, 2-n-heptyl-4-hydroxyquino-line-N-oxide (HQNO), rotenone, flavine adeninedinucleotide, flavine mononucleotide, from SigmaChemical Co., St. Louis, Mo.; D,L-sodium lactate,from Fisher Scientific Co., Pittsburgh, Pa.; phenazinemethosulfate from Mann Research Laboratories, NewYork, N.Y.; 2,4-dinitrophenylhydrazine, 4,4,4-tri-fluoro-l-(2-thienyl)-1, 3-butanedione (TFTB) fromEastman Organic Chemical Co., Rochester, N.Y.;sodium pyruvate, oxaloacetic acid, L(+) lactic acid,D,L-a-hydroxybutyric acid, D(+) malate, menadione(vitamin K3) from Calbiochem, Los Angeles, Calif.;glycollic acid from Matheson Co., East Rutherford,N.J.; chlorpromazine hydrochloride, gift of Smith,Kline, & French, Philadelphia, Pa.

Chemical methods. Pyruvate was identified as theend product of D(-) lactate oxidation by forming(29) and chromatographically characterizing the 2,4-dinitrophenylhydrazine derivative (28) in the con-ventional manner. Reaction-time intervals of 5 to 15min and an enzyme concentration of approximately0.5 mg/ml were used in studies where the keto endproduct of D(-) lactate oxidation was trapped as the2,4-dinitrophenylhydrazone derivative. Studies onreaction stoichiometry that involved the quantitativeoxidation of D(-) lactate as well as the completedecarboxylation of pyruvate required longer time in-tervals and greater enzyme concentrations as indicatedin Table 1.

For use in manometric assays, a 1% solution (0.033M) of phenazine methosulfate was prepared with de-ionized water, and a 2% solution (0.116 M) of men-adione was prepared in 95% ethyl alcohol. Both solu-tions were stored in dark tubes and used within 72 hrafter preparation. Stock solutions of Amytal, anti-mycin A, HQNO, rotenone, and TFTB were pre-pared in 95% ethyl alcohol. A stock solution of dicu-marol was prepared with 0.1 M NaOH; subsequentdilutions were carried out in deionized water. Stocksolutions of chlorpromazine and potassium cyanidewere made in deionized water. Protein was determinedby a modification of the biuret method of Gornall,Bardawill, and David (8).

Preparation ofA. vinelandii 0 cell-free extracts. Thepreparation of the Azotobacter R3 fraction was de-scribed previously (15, 16). In Table 3 are summarizeddata showing specific activities for D(-) lactateoxidation and recovery of protein and the activity

units which resulted from the fractionation proce-dure.Enzyme assays. The D(-) lactic oxidase activity

was assayed manometrically at 37 C by using air asthe gas phase and conventional Warburg vessels of15-ml capacity. The main compartment of the flaskcontained tris(hydroxymethyl)aminomethane (Tris)-chloride buffer (pH 8.5), 33.3 mm; the Azotobacter R3fraction, 0.33 to 0.83 mg of protein per ml; anddeionized water to a total volume of 3.0 ml. Thecenter well contained 0.2 ml of 2 M KOH solution. Theside arm contained a solution of 100 /Amoles ofDL-sodium lactate or 50 ,smoles of D(-) sodiumlactate which was added to start the reaction afterthe initial temperature equilibration period of 7 min.The phenazine methosulfate reductase assay was

identical to that described for the lactic oxidase assay,except that the reaction was assayed with potassiumphosphate buffer (pH 7.0), 33.3 mm; and 3 mM KCN.In this assay, the phenazine methosulfate (0.2 ml of a1% solution) was tipped from a second side arm at thesame time that lactate was added to initiate the reac-tion.

Similarly, for the menadione reductase assay, themain compartment contained Tris-chloride buffer(pH 8.0), 33.3 mm; KCN, 3 mm; and the final concen-tration of the Azotobacter R3 fraction ranged from 0.67to 1.0 mg of protein per ml. Menadione (0.03 ml of a2% solution in 95% ethyl alcohol) was added initiallyto the main compartment. All other features of theassay were identical to that of the oxidase assay.

Specific activities were expressed routinely in micro-liters of 02 uptake per hour per milligram of proteinat 37.C.

Spectral studies. For difference spectra studies (re-duced minus oxidized), the Azotobacter R3 fractionwas suspended in 0.02 M phosphate buffer, pH 7.5, ata concentration of 10 mg of protein per ml. Steady-state reduction was achieved by adding DL-sodiumlactate at a final concentration of 0.33 M. Chemicalreduction was achieved by adding a few crystals ofdithionite.

RESULTS

Stoichiometry of reaction. The oxidation oflactate is carried out by the Azotobacter R3 elec-tron transport fraction according to the followingoverall reaction:

COOH °OOH2 HC--OH + 02 2 =O + 2 HOH

CH3 CH3

Paper chromatography studies on the 2,4-dinitro-phenylhydrazones formed from the end productverified that pyruvate was a major end product ofthe reaction. The 2,4-dinitrophenylhydrazine de-rivative of the unknown end product, as well asthat of pyruvate, a-ketoglutarate, and oxaloace-tate, were resolved and identified by paper chro-matography by using three solvent systems pre-viously described (28).Assuming the complete enzymatic oxidation of

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TABLE 1. Reactioli stoichiometry for the oxidationt of D(-) lactate anld decarboxylation ofpyruvate by the Azotobacter R3 fractioni

02 consumed' (umoles) C02 evolvedh (Gmoles)Substrate (moles/vessel) Theory (%) Theory (%)

Actual Theory Actual Theory

D(-) Lactate 10.0 4.7 5.0 95 9.8 10.0 98Pyruvate 10.0 0.0 0.0 0 9.1 10.0 91

a Protein concentration of the R3 fraction used was 7.3 mg/ml; reaction time was 3 hr.b Protein concentration of the R3 fraction used was 6.8 mg/ml; reaction time was 30 min.

TABLE 2. Comparative studly oli the ability ofAzotobacter R3 Jractioni to oxidize D(-) lactate

and other related a -hydroxyacid substrates

Oxidase activitv

Substrate' ConcnSpecificbaciiy Activity

D(-) Lactate 16.7 244 100D,L-Lactate 33.3 249 102D, L-a-Hydroxy- [ 33.3 198 81

butyrate

,, The following substrates were not oxidized:L(+) lactate (16.7 mM), D,L-ca-hydroxyisobu-tyrate (33.3 mM), D,L-a-hydroxyisovalerate (33.3mM), D, L-a-hydroxycaproate (33.3 mM), glycol-late (33.3 mM), and D(+) malate (33.3 mM).

b Expressed in microliters of 02 uptake perhour per milligram of protein (37 C).

lactate to pyruvate, 2 ,umoles of lactate are oxi-dized by 1 ,umole of oxygen to 2 ,umoles each ofpyruvate and water. This reaction stoichiometrywas readily confirmed (Table 1). For these studies,an excess of the Azotobacter R3 fraction wasadded to the reaction used to insure completeand rapid oxidation of 10 ,umoles of D( -) lactate.Further studies on the reaction stoichiometryrevealed that lactate can also be decarboxylatedby the particulate Azotobacter R3 fraction (Table1). When 10 ,umoles of lactate was allowed to beoxidized completely, 9.8 ,umoles of CO, wasformed. In a similar study, when 10limoles ofpyruvate was used as substrate, there was nooxygen uptake, although 9.1 ,umoles of carbondioxide was liberated after acidification.

This series of studies strongly suggested thatthe electron transport system of A. vinelandiireadily oxidizes lactate to pyruvate which can betrapped as 2,4-dinitrophenylhydrazine derivativePyruvate, in turn, can be further decarboxylatednonoxidatively, indicating the presence of apyruvic decarboxylase in the Azotobacter cell-free extract. Prelimrinary observations indicatedthat C02, acetaldehyde, and acetoin are the prob-

able end products of this nonxidative decarboxy-lation of pyruvate.

Oxidation of D( -) lactic acid and other a-hydroxyacids. The lactic oxidase in the Azotobac-ter R3 fraction also was examined for its ability tooxidize D( -) lactate as well as other a-hydroxy-acid substrates (Table 2) D(-) Lactate wasoxidized readily at a rate of 244 ,uliters of 02consumed per hr per mg of protein, whereas theL(+) isomer was inactive. The racemic mixtureof D,L-lactate was oxidized at a rate of 249 ,ulitersof 02 uptake per hr per mg of protein, a specificactivity equivalent to that observed for the D(-)stereoisomer. Of the series of other a-hydroxy-acid substrates tested, only D,L-a-hydroxybuty-rate was oxidized. The activity noted for this sub-strate represented 81 ' of the activity obtainedfor D(-) lactate. No other a-hydroxyacid testedwas oxidized by the Azotobacter R3 fraction.Glycollate (or a-hydroxyacetate) was not oxi-dized, which indicated that the D(-) lactic oxi-dase of the R2 fraction required at least threecarbons for activity. D(+) Malate, the unnaturalisomer of the acid, also was not oxidized, indi-cating that an addition of a carboxyl group to thethird carbon atom of D( -) a-hydroxyacid rendersthis substrate inactive. The L(-) malate can beoxidized at appreciable rates by the AzotobacterR:t electron transport (4, 14, 20; P. Jurtshuk, C. H.Denton, and A. J. Bednarz, Bacteriol. Proc.,p. 104, 1967).

This same pattern of substrate specificity fora-hydroxyacids is reminiscent of that reportedfor the D( -) lactic cytochrome c reductase fromaerobically grown S. cerevisiae (12). In contrast, ithas been reported (10, 30) that a large numberof D(-) a-hydroxyacids were capable of beingoxidized by the soluble D(-)-a-hydroxyacid de-hydrogenase isolated from anaerobically grownS. cerevisiae.

Effects oJfflavine adenine dinucleotide andflavinemononucleotide On D(-) lactate oxidation. Sinceother known D- and L-lactic acid oxidoreductasesare flavoprotein enzymes, they possess eitherflavine adenine dinucleotide or flavine mononu-

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cleotide as prosthetic groups (3, 11, 25). A studywas undertaken to determine the effect of thesetwo flavine cofactors on D(-) lactate oxidationby the Azotobacter R3 fraction. Neither flavineadenine dinucleotide nor flavine mononucleotidestimulated this oxidation, regardless of the ter-minal acceptor used (phenazine methosulfate,menadione, or molecular 02). No attempt wasmade to identify the types of flavine componentspresent in the Azotobacter R3 fraction, since thespectral characteristics of this fraction revealthe presence of a large concentration of flavopro-tein (16).

Development of assay and enzyme kinetics. Theeffects of various concentrations of D( -) lactateon the activity of the Azotobacter R3 fraction byusing the three different electron acceptors areshown in Fig. 1. This composite figure shows thecomparative activities obtained when the threeelectron acceptors are used as oxidants. Specificactivity is plotted as a function of D(-) lactateconcentration. The highest specific activities andVmax value were obtained when phenazine metho-sulfate was used as electron acceptor. The natural

acceptor, molecular oxygen, also gave a relativelyhigh Vmax value for lactate oxidation, whereasmenadione gave the lowest value. All acceptorsappeared to be saturated at approximately 1.5mM of D(-) lactate. First-order kinetics weredisplayed by the D(-) lactic oxidase system atconcentration levels approaching and slightlymore than the 1.5 mm level of D(-) lactate,whereas the assay with phenazine methosulfateand menadione exhibited first-order kinetics at amuch lower substrate concentration level, ap-proximately 0.75 mm. At higher D(-) lactateconcentrations, the oxidation rate increased onlygradually with increasing substrate concentrationand only for the oxidase and menadione reductaseassays. Analyses by the double-reciprocal plot(insert, Fig. 1) for the oxidase assay gave a K,,value for D(-) lactate of 0.003 M and a Vvalue of 714 ,uliters of 02 uptake per hr per mg ofprotein at 37 C. The phenazine methosulfatereductase had a K,l value for D(-) lactate of0.002 M and a Vn,ax value of 909. The menadionereductase exhibited the lowest K,m value for D(-)lactate, or 0.0008 M and a Vrnax value of 333. The

1.0 " 1.5D-LACTATE (mM)

FIG. 1. Effect of D(-) lactate concentration on the lactic oxidase, phenazine methosulfate reductase, andmenadione reductase in the Azotobacter R3 fraction.

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final D( -) lactate concentration selected for use

in all three assay systems was 0.0167 M. Thisconcentration was approximately 6, 8, and 20times the Knm value for the lactic oxidase, phen-azine methosulfate reductase, and menadionereductase, respectively.

Distribution ofenzymatic activitiesfor D(-) lac-tate oxidation in cell-free extracts. A representa-tive fractionation scheme showing the distributionof activity units for D(-) lactate oxidation incell-free extracts is shown in Table 3. These frac-tions were obtained by differential centrifugationfrom sonically disrupted resting cells of A. vine-landii. The residue fractions are designated "R"and the supernatant fractions are designated "S."The assays developed for the three electron accep-tors (molecular 02, phenazine, methosulfate, andmenadione) were performed on each of the frac-tions. The S, fraction or the first supernatantfraction was collected after centrifugation of thesonic homogenate at 4,300 x g for 10 min. Thisremoved all unbroken whole cells, as well as thelarge cellular debris matter. Centrifugation of theS, fraction at 37, 000 x g for 20 min gave twofractions, the S2 and R2, which contained 80%and 13% protein, respectively. Further centrif-ugation of the S2 fraction at 144,000 X g for2 hr separated the S3 from the R3 fraction. Theformer contained 50%, of the remaining protein,and the latter contained 38 %7. The highest activityfor lactate oxidation was found in the R3 particu-late fraction, which exhibited approximately a

two- to threefold purification when compared tothe original S, extract. The S3 fraction exhibitedlittle or no activity for D(-) lactate oxidation,regardless of the electron acceptor used. All sub-sequent analyses revealed that the greatest num-

ber of activity units were recovered in the particu-late R3 fraction. The percentage of recovery ofenzyme units in the R3 fraction (from the S, frac-tion) was approximately 64% for the D(-) lactic

oxidase, 91% for the phenazine methosulfatereductase assay, and 49%O for the menadionereductase assay.

Difference spectrum of the Azotobacter R3fraction. Studies on the steady-state reduction thatdirectly affects the oxidation-reduction compo-nents of the Azotobacter R3 (Fig. 2) verified thata substantial proportion of the electron transport

600WAVELENGTH (m,e)

FIG. 2. Difference spectrum (reduced minus oxidized)exhibiting the steady-state reduction of the redox com-

ponents in the Azotobacter R3 electron transport frac-tion. Dashed line shows the reduction achieved by theaddition ofD(-) lactate; solid linte represents the reduc-tion by dithionite. Concentrationi of the R3 fraction was

10 mg ofprotein in 1.0 ml of 0.02 M phosphate buffer,pH 7.5.

TABLE 3. Distribution of activity units for D(-) lactate oxidation in fractions obtained bydifferential centrifugation from sonically disrupted resting cells of Azotobacter vinelandii

Specific activitya and recovery of activity units

A otobacter fractions Protein recovery

(%)02 Total unitsb PMS Total units1 K3 Total unitsb

SI 100 91 100 114 100 94 100S2 80 80 70 103 72 95 81R2 13 102 14 191 22 107 15

S2 100 80 100 103 100 95 100S3 50 0 0 39 19 0 0R3 38 193 91 344 126 152 60

a Expressed in microliters of 02 uptake per hour per milligram of protein (37 C).b Calculated by multiplying total protein concentration by specific activity.

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components, viz., the cytochromes and flavo-protein, were reduced by both D(-) lactate anddithionite. Reduction of cytochromes a, + a2(590 and 629 m,u, respectively), cytochromeb, (561 and 531 m,u),and cytochromes C4 + c5 (553and 523 m,u) was readily accomplished by theaddition of lactate. However, a much smallerpercentage of total flavoprotein appeared to bereduced by lactate. The trough at 457 m,u wascharacteristic of bleaching obtained for flavo-protein and possibly nonheme iron reduction.Complete reduction of all the oxidation-reductioncomponents was obtained by the addition of a fewcrystals of sodium dithionite.

Effects of inhibitors. The effects of various elec-tron transport inhibitors on the D( -) lacticoxidase activity in the R3 fraction are shown inFig. 3. Chlorpromazine proved to be the mostpotent inhibitor, 50% inhibition occurring at aconcentration of 6.6 x 10-6 M. Complete inhibi-tion with chlorpromazine was obtained at a con-centration of 1.0 x 10-4 M. Neither the L-malicoxidase nor the succinoxidase were so sensitiveto chlorpromazine. Similar inhibition patternsfor the D(-) lactic oxidase were observed withantimycin A, HQNO, and cyanide. Withantimycin A, 50% inhibition occurred at approxi-mately 5 X 10-5 M; with HQNO and cyanide,50% inhibition occurred at a level of approxi-mately 10-4 M. Comparative studies were madeon the D(-) lactic oxidase and succinoxidase inthe same Azotobacter R3 preparations, and it wasobserved that the latter was consistently 10 timesmore sensitive to HQNO and cyanide. This sug-

gests that there might be two different pathwayswith electron transport chains, one predominantlyfor D(-) lactate oxidation and the other for suc-cinate oxidation.Rotenone showed a peculiar inhibition pattern

which was nonlinear in comparison to the otherinhibititors tested. Extrapolation of the rotenonecurve shows a theoretical 50% inhibition at 2.5x 10-4 M. This unusual type of inhibitor responseby rotenone can perhaps be best explained by itsinsolubility in aqueous systems at high concen-trations.Dicumarol and TFTB appeared to be the poor-

est inhibitors of D( -) lactic oxidase. These inhibi-tors displayed 50% inhibitions at 4.5 X 10-4 Mand 6.0 X 10-4 M, respectively. One interestingaspect of this study was that TFTB, a specific in-hibitor of succinate oxidation, inhibited the D( -)lactic oxidase in the Azotobacter R3 fraction atthe same concentration at which it inhibited suc-cinoxidase.The most effective inhibitors of the D(-) lactic

oxidase activity were chlorpronazine, antimycinA, HQNO, and cyanide.The same electron transport inhibitors (Fig. 3)

were tested for their ability to inhibit the D( -)lactic phenazine methosulfate and menadine re-ductase activities. The concentrations of inhibi-tors used for this study were those which inhibitedthe oxidase by 50%70. Only dicumarol and TFTBinhibited the D(-) lactic phenazine methosulfateand menadione reductase; the inhibition observedwas approximately equal to that noted for theoxidase assay. This suggests that both dicumarol

loN6loT5 IoN4INHIBITOR CONCENTRATION (M)

FIG. 3. Patterns of inhibition observed with various electron transport inhibitors on the D(-) lactic oxidase ofthe Azotobacter R3 fraction.

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and TFTB inhibit the D(-) lactate oxidation ator close to the flavoprotein site, whereas all otherinhibitors function at a site beyond the flavopro-tein region.

DISCUSSIONThere are at least four known classes of flavo-

protein-dependent lactic oxidoreductases thathave been studied in microbial systems. One type,which catalyzes the enzymatic oxidation of theL(+) isomer of lactate, has been characterized inaerobically grown yeast (1) and in the Myco-bacterium sp. (6, 35). In yeast, this enzyme ini-tially concentrates in the particulate fraction and isreferred to as L( +) lactic dehydrogenase(EC 1.1.2.3) or the "cytochrome b2" type prepara-tion. Appleby and Morton (1) were able to crys-tallize this "flavohemoprotein" by use of butanoland acetone fractionation procedures. Its pros-thetic groups were identified as protoheme andflavine mononucleotide. Much less, however,is known about the soluble flavoprotein L-lactic oxidative decarboxylase (EC 1.1.3.2) ofM. phlei (35). This enzyme carries out the directoxidation of L-lactate (by molecular oxygen),forming acetate and CO2. Studies on the crystal-line enzyme have revealed that pyruvate competi-tively inhibits lactate oxidation and that flavinemononucleotide is one of the prosthetic groupspresent on the enzyme.The oxidation of the D(-) stereoisomer of

lactate can occur also via the flavoprotein-typeoxidoreductases. In addition to Azotobacter spp.(4, 14, 20, 38), oxidation specific for the D(-)lactate isomer has been reported in L. mesen-teroides (17), P. pentosaceum (23), and S.cerevisiae (10, 24, 31). Two enzymes of this classhave been examined in microorganisms, i.e., theD(-) lactic cytochrome c reductase (EC 1.1.2.4)and the D(-) a-hydroxyacid dehydrogenase(EC 1.1.99.6). In cell-free homogenates, theformer is found in the particulate fraction ofaerobically grown S. cerevisiae, namely, thathaving electron transport function. The latter,or the D(-) a-hydroxyacid dehydrogenase, hasbeen found in the soluble fraction of anaerobicallygrown yeast (3, 30) as well as in animal tissues(36). Extensive studies on the D(-) lactate cyto-chrome c reductase revealed that it oxidizes solelyD(-) lactate and D(-) a-hydroxybutyrate.The prosthetic group for D(-) lactic cytochromec reductase is flavine adenine dinucleotide, andit also contains bound Zn++, whose concentrationis stoichiometric with the flavine (25, 30). Ithas been referred to as a "metalloflavoprotein"type enzyme. In contrast, the D( -) a-hydroxyaciddehydrogenase is capable of oxidizing most otherD(-) a-hydroxyacids, in addition to D(-) lac-

tate and D( -) a-hydroxybutyrate. The prostheticgroup for the D(-) a-hydroxyacid dehydrogenaseis also flavine adenine dinucleotide, and the puri-fied enzyme is believed to contain divalent metalions (3, 30).The oxidation of D(-) lactate by the Azoto-

bacter R3 fraction exhibits certain similarities anddifferences when compared to the two microbialD(-) lactic oxidoreductases described. The D(-)lactic cytochrome c reductase isolated specificallyfrom aerobically grown S. cerevisiae most closelyresembles the flavoprotein dehydrogenase of A.vinelandii. In both microorganisms, enzymaticactivity is located in the respiratory particlescapable of carrying out electron transport func-tion. Further similarity is noted in the substratespecificity pattern. Of all the a-hydroxyacidstested, both the Azotobacter and Saccharomycesparticulate enzymes were capable of oxidizingonly D(-) lactate and D(-)-a-hydroxybutyrate(Table 2). This specificity suggests that the D(-)lactic oxidase of the Azotobacter R3 fraction is notthe "general" type of D(-)-a-hydroxyacid dehy-drogenase that is found in the soluble fraction inS. cerevisiae grown under anaerobic conditions(3).

Stimulation of D(-) lactate oxidation by eitherflavine adenine dinucleotide or flavine mononucle-otide could not be demonstrated at the level ofpurification achieved for the Azotobacter R3fraction. However, studies on the highly purifiedyeast D( -) lactic cytochrome c reductase ofaerobic yeast indicated that the flavine adeninedinucleotide is the functional flavoprotein moietywhich was reversibly dissociated from the apo-enzyme (12).

Phenazine methosulfate was found to be a goodelectron acceptor for the D(-) lactate oxidationin the Azotobacter R3 fraction; this acceptor alsoserves as a good electron carrier for the purifiedyeast D(-) lactic cytochrome c reductase. TheKm and Vrnax values for phenazine methosulfatereduction with the Azotobacter R3 fraction were0.00015 M and 385 ,uliters of 02 uptake per hr permg of protein, respectively. The latter value cor-responds to 0.578 ,umoles of D( -) lactate oxidizedper min per mg of protein. The correspondingvalues for phenazine methosulfate reduction withthe highly purified yeast D(-) lactic cytochromec reductase were 0.00445 M and 1,670 ,umoles ofD(-) lactate oxidized per min per mg of protein.The comparable yeast particle (from which theenzyme is isolated), or the equivalent of theAzotobacter R3 electron transport fraction, exhib-ited a specific activity of 0.4 umoles of D( -) lactateoxidized per min per mg of protein (12).

In comparing the capability of the AzotobacterR3 fraction to use various electron acceptors for

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D(-) LACTATE OXIDATION IN A. VINELANDII

D(-) lactate oxidation, it was noted that most ofthe electron carriers tested served as acceptors.Menadione, particularly, was found to be aneffective electron acceptor, giving a Vmnax value ofof 1.5 ,umoles of D(-) lactate oxidized per minper mg of protein at infinite menadione concen-tration. In addition to phenazine methosulfateand nrenadione, 2, 6-dichlorophenolindophenol,ferricyanide, and rrethylene blue all served asgood electron acceptors, whereas nitrotetrazoliumblue and cytochrorre c functioned poorly asacceptors. It is probable that this response patternto various electron acceptors will change when theD( -) lactic oxidoreductase of A. vinelandii ispurified to the extent achieved for S. cerevisiae.The sites at which some of these acceptors reactmay be lost or modified on further purification.The specificity for electron acceptors observed

for the Azotobacter R3 electron transport systemis different from that reported for the yeast D( -)lactic cytochrome c reductase (30). Menadione,ferricyanide, 2,6-dichlorophenolindophenol wereall inactive and only phenazine methosulfate andcytochrome c served as electron acceptors forthe purified yeast enzyme. In this respect, theAzotobacter R3 and yeast enzyme systems differedmarkedly. It was observed that, with the exeptionof molecular 02 and phenazine methosulfate, thespecificity for electron acceptors by the Azoto-bacter R3 fraction more closely resembled thatreported for the D(-) a-hydroxyacid dehydro-genase found in anaerobically grown S. cervisiae(30); whereas, with regard to substrate specificity,the Azotobacter R3 system rrore closely resembledthe D(-) lactic cytochrorre c reductase of aero-bically grown yeast.D(-) Lactate oxidation in the Azotobacter R3

fraction appears to be carried out by the electrontransport system. This fact is borne out by theeffect of the various electron transport inhibitorson the D(-) lactic oxidase activity (Fig. 3).Among the most effective inhibitors were anti-mrycin A, HQNO (18), and cyanide, all knowninhibitors of electron transport. The involvenrentof the electron transport system is further sub-stantiated by the data presented in Fig. 2, where-by steady-state reduction studies show that al-n ost all of the oxidation-reduction componentsof the A. vinelandii electron transport chain arereduced by D( -) lactate. This is particularly trueof the cytochrome components (a,, a,, b1, C4, andC5) which are substantially reduced by lactate,when compared to the extent of reductionachieved by dithionite. In contrast, only a smallpercentage of the flavoprotein appears to bereduced by the addition of lactate, suggestingthat the lactic flavoprotein oxidoreductase is

probably present as a minor component in theA. vinelandii R3 electron transport system.One unexpected finding was the sensitivity of

the D(-) lactate oxidation to chlorpromazine.The oxidase activity was inhibited 50%t10 at a6.6 X 10-6 M concentration of chlorpromazine.This concentration level had no effect on the D( -)lactic phenazine methosulfate or menadionereductase activities, thus indicating that it actssomewhere after the flavoprotein site.

ACKNOWLEDGMENTThis investigation was supported by Public Health

Service grant GM-12279 from the National Instituteof General Medical Sciences.

LITERATURE CITED

1. Appleby, C. A., and R. K. Morton. 1959. Lacticdehydrogenase and cytochrome b2 of baker'syeast. Biochem. J. 71:492-499.

2. Asnis, R. E., V. G. Vely, and M. C. Glick. 1956.Some enzymatic activities of a particulate frac-tion from sonic lysates of Eseherichia coli. J.Bacteriol. 72:314-319.

3. Boeri, E., T. Cremona, and T. P. Singer. 1960.The D-a-hydroxyacid dehydrogenase of yeast.Biochem. Biophys. Res. Commun. 2:298-302.

4. Bruemmer, J. H., P. W. Wilson, J. L. Glenn, andF. L. Crane. 1957. Electron transporting particlefrom Azotobacter vinielklidjii. J. Bacteriol. 73:113-116.

5. DeLey, J., and J. Schel. 1959. Studies on the me-tabolism of Acetobacter peroxydauis. II. Theenzymic mechanism of lactate metabolism.Biochim. Biophys. Acta 35:154-165.

6. Edson, N. L. 1951. The intermediary metabolismof the mycobacteria. Bacteriol. Rev. 15:147-182.

7. Eichel, H. J., and L. T. Rem. 1962. Respiratoryenzyme studies in Tetrahymenia pyriformis. V.Some properties of an L-lactic oxidase. J. Biol.Chem. 237:940-945.

8. Gornall, A. G., C. J. Bardawill, and M. M. David.1949. Determination of serum proteins bymeans of the biuret reaction. J. Biol. Chem.177:751-766.

9. Green, M., and P. W. Wilson. 1953. Hydrogenaseand nitrogenase in Azotobacter. J. Bacteriol.65:511-517.

10. Gregolin, C., and T. P. Singer. 1961. The D-lactic cytochrome reductase of yeast: its chem-ical nature, origins, and relation to the respira-tory chain. Biochem. Biophys. Res. Commun.4:189-194.

11. Gregolin, C., and T. P. Singer. 1962. Zinc-FAD,prosthetic groups of D-lactate cytochrome re-ductase. Biochim. Biophys. Acta 57:410-412.

12. Gregolin, C., and T. P. Singer. 1963. D(-) Lacticcytochrome c reductase, a zinc-flavoproteinfrom aerobic yeast. Biochim. Biophys. Acta67:201-218.

13. Haugaard, N. 1959. D- and L-Lactic acid oxidases

VOL. 96, 1968 685

on January 14, 2019 by guesthttp://jb.asm

.org/D

ownloaded from

JURTSHUK AND HARPER

of Escherichia coli. Biochim. Biophys. Acta31:66-72.

14. Jones, C. W., and E. R. Redfearn. 1966. Electrontransport in Azotobacter vinelanzdii. Biochim.Biophys. Acta 113:467-481.

15. Jurtshuk, P., P. R. Aston, and L. Old. 1967.Enzymatic oxidation of tetramethyl-p-phen-ylenediamine and p-phenylenediamine by theelectron transport particulate fraction of Azoto-bacter vinelandii. J. Bacteriol. 93:1069-1078.

16. Jurtshuk, P., and L. Old. 1968. Studies on cyto-chrome c oxidation by the electron transportfraction of Azotobacter vinielanidii. J. Bacteriol.95:1790-1797.

17. Kaufmann, E., and S. Dikstein. 1961. A D-lacticdehydrogenase from Leuconostoc mesenteroides.Nature 190:346.

18. Lightbown, J. W., and F. L. Jackson. 1956.Inhibition of cytochrome systems of heartmuscle and certain bacteria by antagonists ofdihydrostreptomycin: 2-alkyl-4-hydroxyquino-line N-oxides. Biochem. J. 63:130-137.

19. Mahler, H. R., A. Tomisek, and F. M. Huen-nekens. 1953. Studies on the cyclophorasesystem. XXVI. The lactic oxidase. Exptl. CellRes. 4:208-221.

20. Marr, A. G. 1960. Enzyme localization in bac-teria. Ann. Rev. Microbiol. 14:241-260.

21. Millman, I., and G. P. Youmans. 1955. Thecharacterization of the terminal respiratoryenzymes of the H37Ra strain of Mycobacteriumtuberculosis var. hominis. J. Bacteriol. 69:320-325.

22. Mitchell, P., and J. Moyle. 1956. The cytochromesystem in the plasma membrane of membraneof Staphylococcus aureus. Biochem. J. 64:19p.

23. Molinari, R., and F. J. S. Lara. 1960. The lacticdehydrogenase of Propionibacterium pento-saceum. Biochem. J. 75:57-65.

24. Nygaard, A. 1959. Lactic dehydrogenase of yeast.II. Different forms of the enzyme. Biochim.Biophys. Acta 35:212-216.

25. Nygaard, A. P. 1961. D(-) Lactic cytochrome creductase, a flavoprotein from yeast. J. Biol.Chem. 236:920-925.

26. Pangborn, J., A. G. Marr, and S. A. Robrish.1962. Localization of respiratory enzymes inintracytoplasmic membranes of Azotobacteragilis. J. Bacteriol. 84:669-678.

27. Pascal, M. C., and F. Pichinoty. 1963. Stereo-specificite et biosynthese des lactate-dehydro-genases d'Aerobacter aerogenes. Biochim. Bio-phys. Acta 77:507-509.

28. Saini, A. S. 1967. Some technical improvementsin the paper chromatography of keto acid 2,4-dinitrophenylhydrazones. Chromatography 26:539-541.

29. Shriner, R. L., R. C. Fuson, and D. Y. Curtin.1965. The systematic identification of organiccompounds, p. 126. John Wiley and Sons, Inc.,New York.

30. Singer, T. P., C. Gregolin, and T. Cremona. 1963.The nature, mechanism of action, and forma-tion of lactic dehydrogenases in anaerobic andaerobic yeasts, p. 47-79. In B. Wright (ed.),Control mechanisms in respiration and fer-mentation. The Ronald Press Co., New York.

31. Singer, T. P., E. B. Kearney, C. Gregolin, E.Boeri, and M. Rippa. 1960. On the multiplicityof lactic dehydrogenases in yeast. Biochem.Biophys. Res. Commun. 3:428-434.

32. Snoswell, A. M. 1963. Oxidized nicotinamide-adenine dinucleotide-independent lactate dehy-drogenases of Lactobacillus arabinosus 17.5.Biochim. Biophys. Acta 77:7-19.

33. Storck, R., and J. T. Wachsman. 1957. Enzymelocalization in Bacillus megaterium. J. Bacteriol.73:784-790.

34. Straub, F. B. 1940. Crystalline lactic dehydrogen-ase from heart muscle. Biochem. J. 34:483-486.

35. Sutton, W. B. 1957. Mechanism of action andcrystallization of lactic oxidative decarboxylasefrom Mycobacterium phlei. J. Biol. Chem. 226:395-405.

36. Tubbs, P. K., and G. D. Greville. 1961. Theoxidation of D(- )a-hydroxyacids in animaltissue. Biochem. J. 81:104-114.

37. Walker, H., and R. G. Eagon. 1964. Lactic dehy-drogenases of Pseudomonias niatriegens. J. Bac-teriol. 88:25-30.

38. Wilson, T. G. G., and P. W. Wilson. 1955. Theterminal oxidation system of Azotobactervinelandii. J. Bacteriol. 70:30-34.

39. Yamamura, Y., M. Kusunose, and E. Kusunose.1952. Lactic oxidases of Mycobacterium tuber-culosis var. avium. J. Biochem. 39:227-238.

686 J. BACTERIOL.

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.org/D

ownloaded from