Plant Physiol. (1968) 43, 597-605

Respiratory Electron Transport Systems of Aquatic FungiI. Leptomitus lacteus and Apodachlya punctatal

Frank H. GleasonDepartment of Botany, University of California, Berkeley, California 94720

Received Noveember 6, 1967.

Abstract. The electron transport systems of 2 species of aquatic fungi, Leptomitus lacteusand Apodachlya punctata, contained cytochrome a-a3 (605 m,u), 2 b type cytochromes (564 and557 mju), c type cytochrome (551 mg), and flavoprotein, but they appeared to lack cytochromec.. Reduced-minus-oxidized difference spectra and difference spectra in the presence ofantimycin A or cyanide were used to characterize these systems. Studies with the electronmicroscope revealed that hyphae of Leptomitus lacteus contained numerous, oonspicuousmitochondria with tubular cristae.

The respiratory electron transport systems of anumlber of Ascomyeetes and Basidiomycetes havebeen stuldied extensively, but very little is knownabout these systems in Phvcomycetes (15). Alex-opoutllos (2) divided the fungi which were formerlyincluded in the Phycomycetes inito 6 taxonomicgroups (classes). One of these classes is theOomycetes. Two representatives of the Oomycetes,Leptoniitus lacteus and Apodachlya punctata, werechosen for this investigation and both of thesebelong to the order Leptomitalles.

The pioneering work of Schade and Thimanin(20) demonstrated that endogenous respiration inLeptomiiitus was inhibiited by cyanide. Cytochromeswere detected in several genera o,f oomycetes fromthe spectra of reduced cellts observed with a handspectroscope (6). Gleason anid Unestam (13) re-cently found that the endogenous respiration ofLeptomitus, Apodachlya, and Sapromyces wasstrongly inhibifted by both sodium cyanide andantimycin A. Reduced-minus-oxidized differencespectra of homogenized mycelia of these fungi werecob,served at room temperature in a preliminarystudy (13). The purpose of this investigaition wasto study the electron tranisport system's of 2 aerobi,cmem1bers of 'the Leptomitales in more detail and tocompare the resu,lts with daita obtained with otherfungi, animals, and plants.

1 This investigation was supported in part by a UnitedStates Public Health Service Fellowship (;1-F1-GM-20,026-01) and by a National Science Foundation grantto Dr. Roderic B. Park (GB-4245). These data arefrom a thesis presented to the Graduate Division of theUniversity of California in partial fulfillment of therequirements for the doctorate degree.

Materials and Methods

Cultures. Isolates of Leptomitus lacteius (59-5)and Apodachlya punctata (62-4) were kiindly pro-vided by Dr. Ralph Emerson, Departmen't of Botany,University of California, Berkeley.

Growth Media and Cultural Conditions. Thefungi were igrown in broth culture ait 200 on arotary shaker ait 120 rpm for 5 days. The mediumfor Leptomitus contained the following components:glucose 1.0 g, Difco Casamino Acids 2.8 g, DifcoYeast Extract 0.7 g, KHoPO, 1.36 g, Na2HPO40.71 g, MgSO4*7H20 0.12 g, Ca,0l022H,O 0.07 g,FeC13,6H2,0 0.0049 g per liter of gliass double dis-tilled waiter. The medium for Apodachlya con-sisted of glucose 3.0 g, Difco Bacto-Peptone 1.25 gand Difco Yeast Extract 1.25 g per liter and thesame concentration of (salts as in the medium forLeptomitus. One liter of sterille broth was placedin a 24iter Erlenmeyer flask. The salbs and organicconstituents were auttoclaved separately and thencombined after cooling. The initial pH of bothmediia was 6.7. Before growth had stopped, themycelium of each ftungus was collected on cheese-cloth and washed with distilled water at roomtemperature.

Observation of Reduced-Minus-Oxidized Differ-ence Spectra. Intact mycelium from the growthmedium was resuspended in a sma,ll volume of0.25 M sucrose sollution and homogenized by aSorval:l Omnimixer for alboutt 2 minutes whi'le main-tained near 0° with an ice 'bath. The 'density ofthe homogenate was abouwt 20 to 25 mg of mycelium('dry wt) per ml. The hoomogenate was piipetted intotwo 3-m'1 quartz cuvettes. T'he 'difiference spectraat room temperature were recorded between 400 m,u

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and 650 m,u by a Cary 14 split beam spectropho-tometer equlilpped with high intensity light souirce,scatterinig transmission accessory, an(d peni periodconltrol. Ini room temperatture spectra 1 drop of3 % hvdrogen peroxide wxas mixetl into the referencecuivette for oxidati,on of the cytochromes and a fewmilligrams of sodiuim lhydrosuliIfite inlto the samplecuivette fo,r reduction. For endogenotis re(tluctioui,so-diuml suicciniate was a(Ided( to the homogenate in-stea(l of sodiuim hydrosulfite. 'Tlhe reduced miulluoXidized(lifferenice spectrum was recorded severaltiniles to accolun't for noise aind (Irift and was cor-recte(l for baseline absorptioni. 'T'he noise level wasles.s thalni 0.001 optical density uInits at dynode 1andcl pell period controlI position 5.

Spectra were observed at liqlid nitrogeni tem-peratulre wi,th a divilded beam spectrophotometerde-veloped by Chance (9) and adapted for lowtemperature work by Estabrook (10). Reductioniwas always accomplished in the manner describedfor room temperature spectra, but in liquild nitrogenteml)erature spectra, the samples were oxidized bybuibbling oxy-eni for 2 minutes. This procedulresharpncs the peaks and therefore increases thelefilititio of the cytochrome com,ponielnts.

itord(er to distinigiiish b type evtochro,mes frontait type cytochromes, the (liffereuice betweeli

Ilhe ab.sorption o;f homogeniate treated xv ith anlti-tteittl A (2 ,gmo2l), suiccinate, aiitd o\vggei anl(lthat of holmogenaite trea,te(d w ith suiciinate only w\vas-ecor(le(l. A fterx,-ard, both sam,ples x\ ere bubbled

\- itla oxx et nl d the sp)ectrtil w-as recordedi aseeo(Itt tile. .\ntim'cut . itI the presenlce of sti-strate atil oxygetn \\will allow) only the b type cy to-clhronmes to retmiaill cotnpletel\x reduiced (5, 14).

Flecctron Microscopy. I-lyphae of lIeptomlitus(lct(its (59-5) were fix\ed wA-ith 4 % glutaraildehvdefor- 1 and onie-half hottrs, rinsed with buffer, andtheni pustfixed wiAth 2 % osmium tetraoxide it0.1 \t suicrose for 3 houirs at room temperatture.The material wras dehydrated in an acetone seriesfollowed by propyllene oxide. Durinig the dehydra-tion seqtuence, ithe hyphae were post-stained over-iiight w-ith 2 % tiran-l nitrate in 70 % acetoine.After (lehydration, the hyphae were infiltrated witha 1:1 miixture of propylene oxide anid EpoII over-night and then placed in puire EpOIn. The resin waspolymerized for 1 day at 350, for 1 day at 450, andfor 3 (l,ays at 600. The blocks were seotioned witha diamond knife in a Porter-Blum microtome. Thesections required additional staining with lead ci-trate, and then were examined anid photographed ina Siemens Elmiskop-I.

Light Microscopy. Photographs of mitochon-dria in the hyphae o,f Leptomitits were taken witha Zeiss aultomatic "photomicroscope' using a 100 x,lhase contrast, oil immersion objective anid a Zeiss"microflash" attachment.

Isolatioi of 31Mitocliondria. 'T'lhe myceliuim ofLeptomitus lacte7is was stispeinded in a solutioncontaining 0.3 Mr mannitol, 1 ImlM EDTA, and 1 g/l

LU~~~~~~~~~~~L

z

LU ~~~~~~~~~Alz

0 u/ Gt 650

WAVELENGTH (mp)I'il(;. 1. Rle(liuic(lm-iiinus-oxi(liz'e(l difference spectra of

/ Ptotll'tus lactcus (1) anid pod(chlya puntt-ftta (A ) atr-oouit tem1eratiirc. Re(luctioll Wa1t aICcoitlplislied i)v so-dintii li (yrostl fitc. 1) 1solatcd mitoclionldri;t. 2) ltotilogetlize( lly 'celinInl.

bovNiine sertum albumnin at pH 17.2 and(I x-as homnog-enized by a Sorvall. omnimixer as pIrevioously de-scribed. The homogenate was centriftiged for 15minutes at 1000 X g to remove the ce,lll walis andunbroken cells. The precipitate obtained wvas dis-carded, and the supernatant was cenltrifuiged for20 m,inuates at 10,000 X g to sedimenit the mito-chondria. The sedimented mitocohondiria w\ere re-suspended in the same soluitioni that was ulsed forhomogenization and(I were ke,pt at 00 prior to re-cor(ling the reduiced-,minuls-oxidlizedl difference-spectra.

Results

Reduced-Mlinus-Oxidized Difference Spectra.Figure 1 shows ithat the reduced-mintus-oxidizeddifference spectra for homogenized mycelia ofApodachlya and Leptomtitius iare very simila,r. Tihecytochrome systems of both fungi contained thecomponenits listed in table I. Using the procedureof Hackett (14) the total amount of redluciblecytochrome in both fungi was found to be approxi-mately 0.2 pmole/g dry weight (13). However, in

598'

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GLEASON-ELECTRON TRANNSPORI' SYSTEMS OF AQUATrIC FLUNGI

1

Cvytoohrome

a-nn1.

b564

b557c

a

605564557551

Peaks (m,u)

.8l

... ~446529 431525 431521 417

APodachIvl(1 there w-'as proportionately more cyto-chrome c ain;d cytochrome a btut less cytochrome bthan in Leptomnitus. Mitochondria isolaited fromboth fungi cont.ain less cytoch,rome c in proportionto the itotall amouint of cytoch'rome than did homog-enlized cells. A broad trouigh around 465 mu dule toflavoprotein appeared in ithe reduced-minus-oxidizeddifference spectra of both homogenized mycelia andisolated mitochondria. The reduced-mninus-oxidizeddifference of Apodachlva in figutre 2 illustrate theincreased resolution of peaks at liquiid nitrogen

547 Apodachly-

552560 598

AQD. :0.05

H A 2

500 550 600

WAVELENGTH (mp)FIG. 2. Reduced-min-us-oxidized difference spectra

for homogenized mycelium of Apodachlya punctataat liquid nitrogen temperature. 1) Reduction by suc-cinate. 2) Reduction by hydrosulfite.

C,)

z

a-

0

z

'IJ

C-

Apodochlya

605

A*C0.00~

4'jl V

0.05

565

529 557

400 450 500 550 600 650

WAVELENGTH (mp)FIG. 3. Dif ference spectra in the presence of anti-

mycin A for homogenized mycelium. of Apodachlyapunlctata at room temperatuire. In both cases, succinate,antimycin A, and hydrogeni peroxide were added tothe homogenate in the sample absorption cell. In A + Cthe couiten'ts of the reference absorptioni cell wvere re-

dutced bv suicciulate. In B the contents of the referencecell were oxidized by hydrogen peroxide.

temperature. The 2 b type cytobh,rome peaks are

(qite distinct frolm 'the cytochrome c peak. Notethat the wavelength of maximuiim absorption of eachcytochrome has beeln shifted silightly duie to the lowtemperatuire. Also figuire 2 illustrates that thechange in optic-atl density due to redu,ction of cyto-chromes was not as great when the materiaal was

allilowed to become anaerobic with addition of thesubstrate, suiccinate, ;as compared with the sredu,cingagent, hydrosulffite. Non-enzymatically redu,cible b

type cytochrome occurs in the Leptomitales sinceonly aboult one-half of the tota)l b type cytochromewas endogenou,sly 'reducible. Previously, Bonner(5) has reported that non-enzvmatically reduciblecytochrome was present in cauliflower mitochondria.

Antimycin A Difference Spectra. Differencespectra in the presence of an,ti;myciin A were ob-tained wvith Apodachlya 'at room temperature (fig 3)and wiith both Apodachlya and Leptomitus at liquidnitrogen temperature (fig 4). This technique al-lowed ithe identification of 2 b type cytochromes and

I c type cytochrome. Tt is possible that more b and

c type cytochromes were present huit were not re-

solved by these method's. Figure 4 illustrates thatthe same a, b, and c type cytochromes were presentin both Apodachlva and Leptomitus.

Cyanide Difference Spectra. Two types of dif-ference !spectra in the presence of cyanide 'are com-

pared with the reduced-minus-oxidized differencespectrum of Apodachlva punctata in figure 5.Cvtochrome a,., appeared to bind cyanide since the

Table I. Cytochromes Detected in Reduced-Mimus-Oxidized Differenzce Spectra of Leptomitus lacteusand Apodachiva punctata at Rooml Temn peraturc

599

bbl1:1 1.1

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PLANT PHYSIOLOGY

ZI--g'VX O.D.- 0.02

4 ~~~~4C 'sJ 598' -----L

0~

500 550 600WAVELENGTH (mp)

FIG. 4. Reduced-minus-oxidized difference spectra(solid lines) and difference spectra with an,timycin A(broken lines) for homogenized myicelia of Apodachlyapunictata (A), and Leptomnitus lactcus (L), at liquidnitrogen temperature. Reduction was accomplished bvhydrosulfite (solid lines) or by succinate, antimycin A,and oxygen (broken lines). The mycelium in thereference cell was oxidized by bubbling oxygen in bothcases.

a'bsorption peak at 605 mu in reduced celils wasshifted to about 592 m/i with the addition o'f KCN.In addition, when KCN was added to myceliumreduced by 'suecinate, part of the b type cytochromebecame oxidized. This same phenomenon was de-scriibed previously by Bonner (4) in higher plantmitochondria.

The Gross Morphology and Ultrastrutcture ofMitochondria. The hypha of Leptomitus containeda large centrail va,cuole wiith strands of cytoplasmextending around the outside, and these strandswere packed with 'long, conspicuous mitochondria(cf. fig 6). The largest mitochondria were about6 u in length and 1 ,u in diameter. Although thepopu,lation of mito,chondria in Leptomitus w'asheterogeneous wi'th respect to size, this fungu'scontained 'the largest mitochondria that were seenin the order Leptomitales (12). Hyphae of Lep-toinitus were fixed 'and sectioned for o,bservationin the electron microscope. From figure 7, it isevident that the mitochondria of Leptoinitus aresurrounded by a double membrane and con,tainn,umerous tubular oristae.

The high density of xvell-developed mitochhondriain the hypbae of Leptonmitus lacteius suggests thatthis fungus is well celqlipped for an aerobic metabo-lism.

A suspension of mitochondria isolated fromLeptornitus was examined also uinder oil immersiona,t 1000 x w'ith a Zeiss pha,se conitrast microscope.The size and shape of the isolated m,itochondriawere identical 'to those seen in intact hyphae.

Discussion

The endogenous respiration of both Apodachlyaputnctata and Leptomitus lacteuts is strongly inhibitedby antimycin A and 'by sodiulm cyanide (13). Intihe present study, evidence from difference spectraindicate that an'timycin A inhibits electron transportbetween cyto'chrome b an'd c and that cyanide bindsto cytochrome 3. Green algae (22), yeast (1),many h'igher plant tissues (14), and animals (1)are also sensitive t'o these inhibitoors. There are,however, groups o'f closely related microorganismswhich contain both sensitive and resiistan't species(18, 21). In baoteria and bluie green algae o ty'pecyto'c'hromes can futnction as terminal oxidases(3, 7, 8, 23), but Plesnicar et al. (19) have sug-gested that ait least in higher plants o type cy,to-c'hro'mes are not terminal oxi'dases an(d are notresponsible for resi,stance to cyanilde and anitimy-cin A.

Th,e electron transport systems of higher plants(5), some green algae (22), and fungi belongingto the class Oomycetes appear to lack cytochromec1 in contrast to mammals and ibaker's yeast (5, 14,16) w'hich contain this cytochrome. The Oomycetesand the Chloirophyta both contain 2 b type cyto-chromes, but the b type cybtochromes fro'm neithergroutip 'have ibeen purified, and the chemical natureand fuinction of the 2 b type cyto'dhromes are tin-known. The cytochrome U-a., and cytochro'me cfound in Oomycetes appear to be similar to tho'se

zw

a-0

T

Apodachlya AO. D.= 0.02

KCN-Endo

500 550 600 650WAVELENGTH (mp)

FIG. 5. Effects of cyanide on the reduced-minus-oxi-dized difference spectrum for homogenized myceliumof Apodachlya punctata. KCN-Endo (Endogenously re-duced plus KCN) minus (endogenously reduced). Endo-Ox (Endogenously reduced) minus (oxidized). KCN-Ox (Endogenously reduced pllus KCN) minus (oxi-dized).

600

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GLEASON-ELECTRON TRANSPORT SYSTEMS OF AQUATIC FUNGI 601Z i _ ~~~~~~~~~~~~~~~~~... ........... ... ..... '.: .;g,*S_~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~... >. .. .. ..

e :.~~~~~ ~~~~~~~~~~~~~~...:....:

b a M~~~~~~~~~~~~~ E _

.............. F.,

-1110,'izeBC

W.1~~~~~~~~~~~~~~~~

_~~~~~~~g

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~.x.. .......W6~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~.,... ..x.o. @a_ fi :H......%FS

:: C_'....

e_Q

cB~.:........ :: : ,.::..................

.. .... ................................

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GLEASON-ELECTRON TRANSPORT SYSTEMS OF AQUATIC FUNGI

K.4

T

C: :d... :X

.4.

..:...* -sW

.,.:.

FIG. 7. Ultrastructure of mitochondria in Leptoiiitus lacteuts at a magnification of 31,000 X.

Insert

603

76S't-@4i'<ffi>5<' ............ ,^.f. Ni %. .; .............. t >* . > t * A t

j.s ; oj,*: , .. t :*SF. s > .. 8 f'. i i . ; A Yk

.

R.-

.i 1.I.

A.L. A:

tz

I.:

'b'."

4N.

T

Vi

.W,

,oS:

AA

00ANI.-......

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GLEASON-ELECTRON TRANSPORT SYSTEMS OF AQUATIC FUNGI

found in both plants and animals. Not enough isknown albout cytochrome proteins yet for theiir usein the study of evolution and phylogeny in thealgae and in most groups o,f funigi. However, theamino acid sequences and other properties of anumiber of cytochrome c proteins have been deter-mined in many animals as well as a few higherfunigi (11, 17, 24).

No attempt was made in the present study todetect quinones in Oomycetes alithough quiinoneshave been foulnd in other ftungi (15).

Acknowledgments

I am extremely grateful to Dr. Roderic B. Park andto Dr. Walter D. Bonner, Jr. for their encouragementan-d assistance in obtaining room temperature and liquidnitrogen temperature spectra. I alIso thank Dr. RalphEmerson for providing facilities for growing these fungi,Dr. Roderic B. Park for the use of his electron micro-scope, and Dr. Allan C. Wilson for criticism of thismanuscript.

Literature Cited

1. AHMAD, K., H. G. SCHNEIDER, AND F. Ml. STRONG.1950. Studies on the biological action of anti-mycin A. Arch. Biocihem. 28: 2811-94.

2. ALEXOPOULOS, C. S. 1962. Introductory mycology.John WViley and Sons, Inc., New York.

3. ARIMA, K. AND T. OKA. 1965. Cyanide resistancein Achromobacter I. Induced formation of cyto-chrome a2 and its role in cyanide-resistant res-piration. J. Bacteriol. 90: 734-43.

4. BONNER, W. D., JR. .1961. The cytochromes ofplant tissues. In: Haematin Enzymes. J. E. Falk,R. Lemberg, and R. K. Morton, eds. PergamonPress, New York. p 479-500.

5. BONNER, WV. D., JR. 1963. Higher plant cyto-chromes. In: Proceedings of the Fifth Interna-tional Congress of Biochemistry. Vol. II. N. M.Sissakian, ed. Pergamon Press Ltd., Oxford.p 50-62.

6. BOULTER, D. AND E. DERBYSHIRE. 1957. Cyto-dhromes of fungi. J. Exptl. Botany 8: 313-18.

7. BRORERG, P. L. AND L. SMITH. 1966. The cyto-chrome system of Bacillus mnegaterium K M.The presence and some properties of two COl)inding cytochromes. Biochem. Biophys. Acta131: 479-89.

8. CASTOR, L. AND B. CHANCE. 1959. Photochemicaldeterminations of the oxidase of bacteria. J.Biol. Chem. 234: 1587-92.

9. CHANCE, B. 1957. Techniques for the assay ofrespiratory enzymes. In: Methods in Enzymology.Vol. IV. S. P. Colowick and N. 0. Kaplan, eds.Academic Press, New York. 273-329.

10. ESTABROOK, R. W. 1956. The low temperaturespectra of hemoproteins. I. Apparatus and itsapplication to a study of cytochrome c. J. Biol.Ghem. 223: 781-94.

11. FITCH, W. M. AND E. MARGOLIASH. 1967. Con-struction of phylogenetic trees. Science 155:279-84.

12. GLEASON, F. H. 1967. Nutrition, fermentation,and terminal respiration in the Leptomitales. Ph.D.Thesis, University of California, Berkeley, Cali-fornia.

13. GLEASON, F. H. AND T. UNESTAMI. 1968. CO11-parative physiology of respiration in aquaticfungi. I. The Leptomitales. Physiol. Plantarum21: 556-72.

14. HACKETT, D. P. 1964. Enzymes of terminal res-piration. In: Modern iAIethods of Plant Analysis.Vol. 7. K. Paech, B. D. Sanwal, and M. V.Tracey, eds. Springer Verlag, Berlin. p 647-94.

15. LINDENMAYER, A. 1965. Carbohydrate metabolism3. Terminal oxidation and electron transport. In:The Fungi. Vol. I. G. C. Ainsworth and A. S.Sussman, eds. Academic Press, New York. p301-48.

16. MACKLER, B. AND H. M. DUNCAN. 1967. Electrointransport systems of yeast. IV. Preparation andproperties of a particulate DPNH, succinate cyto-chrome c reductase. Arch. Biochem. Biophys.118: 542-48.

17. MARGOLIASII, E. AND A. SCHEJTER. 1966. Cyto-chrome c. In: Advances in Protein Chemistry.Vol. 21. C. B. Anfinsen, Jr., M. L. Anson, J. T.Edsall, and F. M. Richards, eds. Academic Press,New York. p 113-286.

18. MIATSUNAKA, S., S. MORITA, AND S. F. CONTI.1966. Respiratory system of Rhodotorula glutinis.I. Inhibitor tolerance and cytoohrome components.Plant Physiol. 41: 1364-69.

19. PLESNICAR, M., XVr. D. BONNER, JR., AND B. T.STOREY. 1967. Peroxidase associated with higherplant mitochondria. Plant Physiol. 42: 366-70.

20. SCHADE, A. L. AND K. V. THIMANN. 1940. Themetabolism of the water mold Leptomlitus lacteus.Am. J. Botany 27: 659-70.

21. UNESTAM, T. AND F. H. GLEASON. 1968. COM-parative physiology of respiration in aquatic fungi.II. Saprolegniales, especially Aphanontyces as-taci. Physiol. Plantarum 21: 573-88.

22. WEBSTER, D. A. AND D. P. HACKETT. 1965. Res-piratory chain of colorless algae. I. Chlorophytaanld Euglenophyta. Plant Physiol. 40: 1091-1100.

23. WEBSTER, D. A. AND D. P. HACKETT. 1966. Res-piratory dhain of colorless algae. II. Cyanophyta.Plant Physiol. 41: 599-605.

24. YAMANAKA, T. AND K. OKUNUKI. 1964. Com-parative biochemistry of cytochrome c. J. Biol.Chem. 239: 1813-17.

605

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