JOURNAL OF BACTERIOLOGY Vol. 88, No. 1, p. 122-129 July, 1964 Copyright ) 1964 American Society for Microbiology Printed in U.S.A. RESPIRATORY PATHWAYS IN THE MYCOPLASMA II. PATHWAY OF ELECTRON TRANSPORT DURING OXIDATION OF REDUCED NICOTINAMIDE ADENINE DINUCLEOTIDE BY MYCOPLASMA HOMINIS P. J. \ANDEMARK1 AND P. F. SMITH Department of Microbiology, School of Medicine, University of South Dakota, Vermillion, South Dakota Received for publication 26 February 1964 ABSTRACT VANDEMARK, P. J. (University of South I)a- kota, Vermillion), AND P. F. SMITH. Respiratory pathways in the Mycoplasma. II. Pathway of electron transport during oxidation of reduced nicotinamide adenine dinucleotide by Mycoplasma hominis. J. Bacteriol. 88:122-129. 1964.-Unlike the flavin-terminated respiratory pathway of the fermentative Mycoplasma, the respiratory chain of the nonfermentative M. hominis strain 07 ap- pears to be more complex, involving quinones and cytochromes in addition to flavins. In addition to reduction by reduced nicotine adenine dinucleo- tide (NADH) and reduced nicotine adenine dinu- cleotide phosphate, nonpyridine nucleotide- linked reduction of the respiratory chain of this organism occurred with suceinate, lactate, and short-chained acyl coenzyme A derivatives as electron donors. Enzymes catalyzing the oxi- dation of NADH included an NADH oxidase, a diaphorase, a quinone reductase, and a cyto- chrome c reductase. The oxidation of NADH was sensitive to a variety of inhibitors, including 10-4 M Atabrine, 10-3 M sodium amytal, 10-5 M p-chloromercuribenzoate, 10-4 M antimycin A, and 10-4 M potassium cyanide. The oxidase was resolved by the addition of 5% trichloroacetic acid and reactivated by the addition of flavin adenine dinucleotide but not flavin mononucleo- tide. The M. hominis sonic extract contained an NADH-coenzyme Q reductase. The oxidation of NADH was stimulated by the addition of either menadione or vitamin K2 (C35). The oxidase was inactivated by extraction with ether or irradiation at 360 m,. The ether-inactivated enzyme was partially reactivated by the addition of "lipid" extract of the enzyme and coenzyme Q6. l)iffer- ence spectra of the cell extracts revealed the presence of "b" and "a" type cytochromes. These cell extracts were found to contain a cyanide- and azide-sensitive cytochrome oxidase and catalase. 1 Present address: Division of Bacteriology, New York State College of Agriculture, Cornell University, Ithaca, N.Y. Previous studies of the respiratory mecha- nisms in the Mycoplasma would indicate that these organisms possess a flavin-ternminated re- spiratory chain and lack the heme-containing respiratory enzymes, e.g., catalase and cyto- chromes (Pirie, 1938; Kandler and Kandler, 1955; Kandler, Zehender, and Miller, 1956; Weibull and Hammarberg, 1962; Smith, Van- Demark, and Fabricant, 1963). However, the majority of these studies have been limited to a relatively few, primarily fermentative, strains of this groul) of organisms. Furthermore, because fermentative ilycoplasma strains possess a glyco- lytic-type metabolism (Rodwell and Rodwell, 1954; Tourtellotte and Jacobs, 1960; Gill, 1960; Castrejon-Diez, Fisher, and Fisher, 1963), the primary electron donors of their respiratory chain would appear to be the reduced pyridine nucleotides. In contrast to the above investigations, Lecce and Morton (1954), studying three human strains of Mfycoplasma, reported the presence of catalase and a cyanide-sensitive respiration as well as a lactate oxidase not linked to pyridine nucleotide. The present study with .I. hominis strain 07 confirms the latter investigation, and indicates that the respiratory chain of this nonfermentative strain is relatively complex, with the oxidation of the reduced pyridine nucleotides involving quinones, eytochromes, and cytochrome oxidase in addition to the flavins. Although the present report will concern itself primarily with the pathway of reduced nicotine adenine dinucleotide (NADH) oxidation, coenzyme A and its acyl derivatives (Acyl-CoA) and suceinate, as well as lactate, appear to serve as electron donors of the respiratory chain of this .7fycoplasma. MATERIALS AND METHODS Cultures. Ml. hominis 07 was grown and har- vested as previously described (Smith, 1955) ex- 122 on April 17, 2020 by guest http://jb.asm.org/ Downloaded from
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JOURNAL OF BACTERIOLOGYVol. 88, No. 1, p. 122-129 July, 1964Copyright ) 1964 American Society for Microbiology
Printed in U.S.A.
RESPIRATORY PATHWAYS IN THE MYCOPLASMAII. PATHWAY OF ELECTRON TRANSPORT DURING OXIDATION OF REDUCED
NICOTINAMIDE ADENINE DINUCLEOTIDE BY MYCOPLASMA HOMINISP. J. \ANDEMARK1 AND P. F. SMITH
Department of Microbiology, School of Medicine, University of South Dakota, Vermillion, South Dakota
Received for publication 26 February 1964
ABSTRACT
VANDEMARK, P. J. (University of South I)a-kota, Vermillion), AND P. F. SMITH. Respiratorypathways in the Mycoplasma. II. Pathway ofelectron transport during oxidation of reducednicotinamide adenine dinucleotide by Mycoplasmahominis. J. Bacteriol. 88:122-129. 1964.-Unlikethe flavin-terminated respiratory pathway of thefermentative Mycoplasma, the respiratory chainof the nonfermentative M. hominis strain 07 ap-pears to be more complex, involving quinonesand cytochromes in addition to flavins. In additionto reduction by reduced nicotine adenine dinucleo-tide (NADH) and reduced nicotine adenine dinu-cleotide phosphate, nonpyridine nucleotide-linked reduction of the respiratory chain of thisorganism occurred with suceinate, lactate, andshort-chained acyl coenzyme A derivatives aselectron donors. Enzymes catalyzing the oxi-dation of NADH included an NADH oxidase,a diaphorase, a quinone reductase, and a cyto-chrome c reductase. The oxidation of NADH wassensitive to a variety of inhibitors, including10-4 M Atabrine, 10-3 M sodium amytal, 10-5 Mp-chloromercuribenzoate, 10-4 M antimycin A,and 10-4 M potassium cyanide. The oxidase wasresolved by the addition of 5% trichloroaceticacid and reactivated by the addition of flavinadenine dinucleotide but not flavin mononucleo-tide. The M. hominis sonic extract contained anNADH-coenzyme Q reductase. The oxidation ofNADH was stimulated by the addition of eithermenadione or vitamin K2 (C35). The oxidase wasinactivated by extraction with ether or irradiationat 360 m,. The ether-inactivated enzyme waspartially reactivated by the addition of "lipid"extract of the enzyme and coenzyme Q6. l)iffer-ence spectra of the cell extracts revealed thepresence of "b" and "a" type cytochromes. Thesecell extracts were found to contain a cyanide-and azide-sensitive cytochrome oxidase andcatalase.
1 Present address: Division of Bacteriology,New York State College of Agriculture, CornellUniversity, Ithaca, N.Y.
Previous studies of the respiratory mecha-nisms in the Mycoplasma would indicate thatthese organisms possess a flavin-ternminated re-spiratory chain and lack the heme-containingrespiratory enzymes, e.g., catalase and cyto-chromes (Pirie, 1938; Kandler and Kandler,1955; Kandler, Zehender, and Miller, 1956;Weibull and Hammarberg, 1962; Smith, Van-Demark, and Fabricant, 1963). However, themajority of these studies have been limited to arelatively few, primarily fermentative, strainsof this groul) of organisms. Furthermore, becausefermentative ilycoplasma strains possess a glyco-lytic-type metabolism (Rodwell and Rodwell,1954; Tourtellotte and Jacobs, 1960; Gill, 1960;Castrejon-Diez, Fisher, and Fisher, 1963), theprimary electron donors of their respiratorychain would appear to be the reduced pyridinenucleotides.
In contrast to the above investigations, Lecceand Morton (1954), studying three humanstrains of Mfycoplasma, reported the presence ofcatalase and a cyanide-sensitive respiration aswell as a lactate oxidase not linked to pyridinenucleotide.The present study with .I. hominis strain 07
confirms the latter investigation, and indicatesthat the respiratory chain of this nonfermentativestrain is relatively complex, with the oxidationof the reduced pyridine nucleotides involvingquinones, eytochromes, and cytochrome oxidasein addition to the flavins. Although the presentreport will concern itself primarily with thepathway of reduced nicotine adenine dinucleotide(NADH) oxidation, coenzyme A and its acylderivatives (Acyl-CoA) and suceinate, as well aslactate, appear to serve as electron donors of therespiratory chain of this .7fycoplasma.
MATERIALS AND METHODS
Cultures. Ml. hominis 07 was grown and har-vested as previously described (Smith, 1955) ex-
cept for the addition of 0.5% sodium acetate tothe culture medium.
Enzymatic methods. Cell-free extracts wereprepared by sonic oscillation of resting-cell sus-pensions in a 10-kc Raytheon oscillator for 10min. The cell debris was removed by centrifuga-tion at 30,000 X g for 30 min, and the resultingsupernatant fraction was used for enzymaticanalysis. The protein level of cell extracts wasdetermined by the trichloroacetic acid methodof Stadtman, Novelli, and Lipmann (1951).The NADH oxidase and menadione reductase
were measured spectrophotometrically as thechange in absorbancy at 340 m,u, as previouslydescribed (Smith et al., 1963). Dichlorophenolindophenol (DCPIP) reduction was measuredas the decrease in absorbancy at 600 m,, andcytochrome c as the increase in optical densityat 550 mg.The oxidation of butyryl-CoA, succinate, and
lactate was assayed according to the method ofHauge (1956), with the use of a comnbination ofphenazine methosulfate and DCPIP, and bymeasuring the decrease in absorption at 600 m,.The flavin prosthetic group of NADH oxidase
was split off from the apoenzyme under acidconditions with either acid ammonium sulfateat pH 2, by the method of Warburg and Christian(1938), or with 5% trichloroacetic acid, as de-scribed by deBernard (1957).The reduction of coenzyme Q was measured
according to the method of Green, Hatefi, andFechner (1959), by which quinone was extractedwith cyclohexane from samples acidified with 1.0ml of 0.1 M perchloric acid. The level of reducedquinone was measured at 275 m,u before andafter complete reduction with sodium boro-hydride.
For studies of the effect of ether extraction onthe NADH oxidase, 1 volume of enzyme wasextracted twice with 10 volumes of diethyl etherat 0 C. The ether extract was evaporated to dry-ness under a stream of nitrogen, and the residuewas redissolved in a small volume of absolutealcohol. This material, necessary for the reactiva-tion of the extracted oxidase, is referred to as the"lipid" extract.
Irradiation of the sonic extract at 360 mu wascarried out according to the method of Brodie,Weber, and Gray (1957).The difference in absorption spectrum between
the sonic preparation in the anaerobic and aerobic
states was measured according to the methodof Wood and Schwerdt (1953) in a Beckmanmodel DU spectrophotometer. The cuvettescontained 3.0 ml of the whole sonic preparationto which was added sodium deoxycholate (1mg/nil). Oxidized conditions were achieved bythe addition of 0.1 ml of 1% potassium ferri-cyanide, and the enzyme was reduced by theaddition of a few crystals of sodium hydrosulfite.
Materials. The vitamin K2 (C35) was kindlysupplied by 0. Isler, Hoffmann-La Rocheand Co., Ltd., Basel, Switzerland. NADH, re-duced nicotine adenine dinucleotide phosphate(NADPH), flavin mononucleotide (FMN), co-enzyme Q6, cytochrome c (horse heart), andphenazine methosulfate were purchased fromSigma Chemical Co., St. Louis, Mo. Flavinadenide dinucleotide (FAD) was purchased fromCalbiochem, and 2,3-dimercaptopropanol (BAL)from Hynson, Westcott & Dunning, Inc., Balti-more, Md. All other chemicals were commercialpreparations of reagent grade.
RESULTS AND DIscussION
Several substrates appear to reduce the re-spiratory chain of nonfermentative M. hominis07 via nonpyridine nucleotide-linked pathways.Butyryl-CoA, succinate, and lactate, in additionto NADH and NADPH, serve as electron donorsof the respiratory chain of this organism (Fig. 1).Succinate oxidation as part of a tricarboxylicacid cycle in this strain was recently observed(VanDemark and Smith, unpublished data). Thenonpyridine-linked butyryl-CoA dehydrogenasewill be reported as part of a study of fatty acidoxidation in this organism (VanDemark andSmith, in preparation). The present report willbe limited primarily to the pathway of NADHoxidation by this strain.Crude extracts of M. hominis 07 were found
to oxidize NADH with oxygen, DCPIP, mena-dione, and cytochrome c as final electron ac-ceptors (Fig. 2). This would suggest the presenceof an NADH oxidase, diaphorase, quinone re-ductase, and cytochrome c reductase in thesecell-free extracts. The crude sonic preparationsoxidized NADPH with the various acceptorslisted in Fig. 2 at approximately one-half therate of NADH oxidation. The actual demonstra-tion of these enzymatic activities as separateentities will depend upon their purification andindividual isolation. Unfortunately, the problems
FIG. 1. Electron donors of the respiratory chain ofMycoplasma hominis 07. The oxidation of NADHand NADPH was followed by the decrease in ab-sorption at 340 mMA (X), and the oxidation of butyryl-CoA, succinate, and lactate as the decrease in opticaldensity at 600 m,u (0). Each cuvette contained 100,umoles of tris(hydroxymethyl)aminomethane buffer(pH 7.4), approximately 1 mg of enzyme protein,and, where indicated, 1 mg of NADH or NADPH,or 10 gimoles of butyryl-CoA, succinate, or lactate,and water to a final volume of 3.0 ml. The cuvettescontaining butyryl-CoA, succinate, and lactate alsocontained 2 mg of phenazine methosulfate and 0.15,umole of DCPIP.
and costs of the mass culture of this strain ofMycoplasma necessary for enzyme purificationmake such studies impractical at this time.
Inhibitor studies. The inhibitor data in Table1 are indicative of a complex pathway of electrontransport occurring during NADH oxidationby this organism. The marked inhibition byAtabrine is evidence for the role of flavins. Sodiumamytal, of which the site of inhibition was shownto be the reduction of the benzoquinones, e.g.,
coenzyme Q (Hatefi et al., 1959), also blocksNADH oxidation by M1. hominis. The oxidationis also sensitive to antimycin A and BAL, chemi-cals known to block the oxidation of reducedcoenzyme Q (Redfearn and Pumphrey, 1959;Green et al., 1959). The inhibition of NADHoxidation of strain 07 by cyanide is characteristicof a cytochrome oxidase-terminated respiration.This pattern of inhibitor sensitivity is presump-
0)
C]0
/ 2.0
9
0
0
t(ID
,YGEN
2
TIME (MINUTES)
FIG. 2. Evidence for an NADH oxidase, quinonereductase, diaphorase, and cytochrome c reductasein Mycoplasma hominis 07. In plot A, the oxidationof NADH was followed as the decrease in opticaldensity at 340 m,; in plot B, the reduction of DCPIP(0) was followed by the decrease in absorption at600 m,, and cytochrome c reduction as the increasein optical density at 550 mM. The cuvettes contained100 umoles of tris(hydroxymethyl)aminomethanebuffer (pH 7.4), approximately 1 mg of enzymeprotein, 1 mg of NADH, and 0.15 ,umole of DCPIP;0.4 Mmole of menadione or 0.15 ,umole of cytochromec was added where indicated. In measuring cyto-chrome c reduction, 0.5 psmole of KCN was addedto the enzyme-buffer mixture 5 min prior to startingthe reaction to inhibit the oxidation of the reducedcytochrome c.
TABLE 1. Effect of some inhibitors on NADHoxidation by Mycoplasma hominis strain 07
Percentage inhibition
Inhibitor*Oxidase Diaphor- Menadione
xae ase reductase
Atabrine (2 X 10-4 M). 70 43 63Sodium amytal (2 X
10-s M) ............. 56 NRt 452, 3-Dimercaptopro-panol (3 X 10-3 M).. 50 NR 20
p-Chloromercuriben-zoate (1.5 X 10-6 M). 62 48 64
Antimycin A (50 ,g percuvette) ............ 50 19 28
KCN (3.3 X 10-4 M) ... 50 5 11
* All inhibitors were incubated with the enzymefor 5 min prior to starting the reaction.
t NR = not run because of endogenous reduc-tion of the dichlorophenol indophenol by the in-hibitor.
tive evidence for a more complex pathway ofelectron transport in this nonfermentative strainthan in the flavin-terminated respiration charac-teristic of fermentative Mycoplasma. This path-way appears to involve quinones and cytochromesas electron carriers.
Flavin requirement ofNADH oxidase. The flavinprosthetic group of the NADH oxidase could besplit from the apoenzyme under acid conditions,with either an acid ammonium sulfate precipita-tion or by the addition of 1 volume of 5% tri-chloroacetic acid. The addition of FAD wasrequired for oxidase activity after this resolution(Fig. 3). However, diaphorase activity was re-activated by the addition of either FAD orFMN, whereas the resolved menadione reductasewas reactivated by the addition of FMN.
Evidence for quinones and naphthoquinones inthe respiratory chain. The addition of coenzymeQ6 did not stimulate the oxidation of NADHby sonic extracts. However, with the techniqueof Green et al. (1959), it was possible to demon-strate the reduction of added coenzyme Q6 bythe cell-free extracts. Quinone is reduced withboth NADH and succinate as electron donors
4
Uc4M FAD
0o 7*
,NO FLAVIN
0 1 2 3 4 5
TIME (MINUTES)
FIG. 3. Flavin requirement of the NADH oxi-dase by Mycoplasma hominis 07. Each cuvettecontained 100 1.Amoles of tris(hydroxymethyl)amino-methane buffer (pH 7.4), 1.6 mg of trichloroacetic-resolved enzyme, 1 mg of NADH, and, where indi-cated, 0.3 Amole of FAD or FMN, and water to a
final volume of 3.0 ml.
0LL 0
( 2P. 6
~~~32~~~
TIME (MINUTES)FIG. 4. Reduction of coenzyme Q6 by Mycoplasma
hominis 07. The reaction was followed accord-ing to the method of Green et al. (1959), with eachtube containing 10jmoles of tris(hydroxymethyl)-aminomethane buffer (pH 7.4), 2 jAmoles of potassiumphosphate (pH 7.4), 6 1Amoles of KCN, 1.2 mg ofenzyme protein, 0.2 mg of coenzyme Q6, and, asindicated, 5 Mmoles of succinate or 0.6 mg of NADH.The reaction was stopped by the addition of 1 ml of0.1 M perchloric acid, and the coenzyme Q6 wasextracted with 3 ml of cyclohexane. The percentageof coenzyme Q6 was determined according to themethod of Redfearn and Pumphrey (1960).
(Fig. 4). This is analogous to the findings ofvarious workers (Hatefi et al., 1959; Ziegler andDoeg, 1959), but differs from the findings ofKashket and Brodie (1963) on quinone reductionby cell-free extracts of Escherichia coli. Theselatter workers observed the reduction of en-dogenous benzoquinone with succinate, but notwith the NAD-linked substrate malate. Thisdifference may be due to the unfavorable reac-tion equilibrium of malate dehydrogenase, re-sulting in only limited levels of NADH beingavailable for quinone reduction.
Organic solvents were used by several workers(Lester, Smith, and Fleischer, 1960; Redfearn,Pumphrey, and Finn, 1960) for the extraction oflipid substances, including the quinones, fromsuccinic oxidase, resulting in the loss of thisenzymatic activity. These extracted enzymescan be reactivated by the addition of benzo-quinone and a portion of the extracted material,which is assumed to be lipid in nature (Redfearnet al., 1960). Extraction of the M. hominis en-
zyme with diethyl ether inactivates the NADHoxidase of this preparation (Fig. 5). This oxidasewas partially reactivated by the addition of co-enzyme Q6 and the "lipid" extract.
Because coenzyme Q6 was the only homologueof this benzoquinone available to us at the time /of this study. we have no data concerning the /effect of the number of isoprenoid units on the K3 (MENADIONE)rate of NADH oxidation. Nor do we have anyevidence concerning the essential components ofthe "lipid" fraction stimulating this oxidase. The /studies of Green and Fleischer (1963) demon- Qstrated the essential role of phospholipids in the °respiratory activity of extracted mitochondria.The cells of M. hominis 07 do contain phospha- /tidylcholine derived from the culture medium K2-(C(d(P. F. Smith, unpublished data), and the possi- 0 o /bility arises that this may be the active compo-nent in the "lipid" extract for NADH oxidation,analogous to the function of phospholipids in the K Kmitochondrial membrane. /
OC., CoQ
0.12 TIME (MINUTES)
/FIG. 6. Relative stimulation of NADH oxidationby the homologues of vitamin K and coenzyme Q6.The cuvettes contained 100 ,moles of tris(hydroxy-
UNTREATED methyl)aminomethane buffer (pH 7.4), 1 mg ofoE ~o ENZYME enzyme protein, 1 mg of NADH, and the variousVI
on # /forms of vitamin K or coenzyme Q6 as indicated.All values are corrected for endogenous NADH
PLUS 'LIPID" oxidase activity.EXTRACT AND
20D AG Co Qo The ability of menadione to serve as an ac-<
O0.4 X / ceptor for NADH oxidation may indicate that a0.04 / /naphthoquinone is functioning in the electron
transport of M. hominis. Figure 6 shows theability of various homologues of vitamin K and
ETHER EXTR'D coenzyme Q6 to stimulate the oxidation ofENZYME
NADH. Of the compounds tested, only vitaminK2 (35) and menadione (K3) resulted in a rate of
0 2 4 6 8 NADH disappearance greater than that ofTIME (MINUTES) NADH oxidase alone.
FIG. 5. Lipid requirements for NADH oxidation Irradiation at 360 m,u with a technique siniilarby ether-extracted Mycoplasma hominis enzyme. to that of Brodie et al. (1957) or Dallam andThe cuvettes contained 100j,moles of tris(hydroxy- Anderson (1957) was found to inactivate NADHmethyl)aminomethane buffer (pH 7.4), 1 mg of oxidase. NADH oxidation by these irradiatedenzyme protein, 1 mg of NADH, and, where indi- preparations occurred in the presence of vitamincated, 0.1 ml of "lipid" extract and 20 ug of coen-zyme Q6. The enzyme was extracted and the "lipid K2 (35) or menadione. However, because naph-extract was prepared as described in Materials and thoquinone stimulated NADH oxidation prior toMethods. irradiation, one cannot conclude that our data
indicate that these naphthoquinones are func-tioning in NADH oxidation.Although the foregoing data would indicate 1.e +3X IeM AZIDE
that both quinones and naphthoquinones canserve as electron acceptors in NADH oxidationby strain 07, the determination of which of these A xcofactors is actually functioning in vivo is yet o 1.6to be made.
Cell extracts of M. hominis 07 gave a positive \test with the Dam-Karrer assay. This colori- NO AZIDEmetric assay, although not highly specific, indi- /cates that these extracts contain a quinone or Clquinones with an unsaturated side chain(s). Weare attempting to identify positively thesequinonelike structures in M. hominis 07. 12
Evidence for catalase and the cytochroms. Lecce 1.and Morton (1954) previously reported that M.hominis strains contained catalase and that theirrespiration was cyanide-sensitive. With thespectrophotometric method of Chance (1949), '°o 2 4
TIME (MINUTES)FIG. 8. Cytochrome oxidase activity of Myco-
0.3/ plasma hominis 07. The cuvettes contained 250Mgmoles of phosphate buffer (pH 7.0), 0.015;Mmole ofreduced cytochrome c (prepared according to Smith,1954), 2 mg of enzyme protein, and, where indicated,1 MAmole of sodium azide.
NO KCN
we demonstrated the presence of this enzyme02/ in the cell-free extracts of M. hominis 07. This
catalase activity and its sensitivity to cyanide0lw / / are illustrated in Fig. 7.
A difference spectrum (aerobic vs. anaerobic)of the whole sonic preparation was made with a
c] ,# / modification of the method of Wood ando 0.1 5XI& M KCN Schwerdt (1953). Absorption bands in the re-
duced state were observed at 565, 532, and 429 mjA,characteristic of a "b" type cytochrome, and at610 and 456 mu, typical of an "a" type cyto-chrome.The presence of cytochrome oxidase was
demonstrated with the technique of Smith (1954).
TIME (MINUTES) Figure 8 illustrates this cytochrome oxidase andits sensitivity to azide. No data concerning the
FIG. 7. Catalase activity of Mycoplasma hominis- nature and role of metals (e.g., iron and copper)strain 07. Enzyme activity was determined spectro, in this oxidase or the other respiratory enzymesphotometrically by the method of Chance (1949), of M. hominis are as yet available.with each cuvette containing approximately 50 The significance of this pathway of electronj.moles of hydrogen peroxide in 2.5 ml of 0.067 M . . .
qphosphate buffer (pH 7), 2 mg of NADH, 0.6 mg of transport, ivolvng fiavins, quiones, and cyto-enzyme protein, and, where indicated, 1.5 ;moles of chromes, is the energy that it can provide thispotassium cyanide (incubated with the enzyme S Mycoplasma. When coupled with the tricar-min before starting the reaction). boxylic acid cycle of this organism (VanDemark
and Smith, unpublished data), the respiratorychain would enable this organism to gain con-siderably more energy from the dissinmilation of asubstrate (e.g., a fatty acid) via oxidative phos-phorylation than from the substrate phosphoryla-tion available during the catabolism of certainamino acids (Smith, 1960; Schimke and Barile,1963). Thus, although both pathways mayprovide energy to this strain, it would seem thatthe oxidative dissimilation of fatty acids wouldbe more expedient as an energy source.
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