Smith College, Northampton, Massachusetts, and - Plant Physiology

PARTICIPATION OF CYTOCHROMES IN THE RESPIRATIONOF THE AROID SPADIX'12

CONRAD S. YOCUM 3 AND DAVID P. HACKETT 4THE BIOLOGICAL LABORATORIES, HARVARD UNIVERSITY, CAMBRIDGE 38, MASSACHUSETTS

Although a cyanide-resistant fraction of respirationis present in many plant and animal tissues, the mech-anism of terminal oxidation in such respiration hasnever been completely clarified. On the basis of theirstudy of the rapid, cyanide-resistant respiration of theAroid spadix, James and Beevers (8) proposed thatflavoproteins serve as terminal oxidases in this tissue.Their conclusion was based in part on the finding thatthe measured respiratory rate was strongly dependentupon the partial pressure of oxygen. In addition,they found no spectroscopic or enzymatic evidencefor the presence of a cytochrome system.

The rapid respiration of the Aroid spadix has beenreinvestigated here. A relative insensitivity to cya-nide (HCN) and carbon monoxide (CO) has beenconfirmed. With an adequate rate of oxygen supply,the tissue exhibits a very high affinity for oxygen.This observation led to a spectroscopic and enzymaticsearch for cytochrome components. The results re-ported here demonstrate the participation of cyto-chromes in the respiration of the Aroid spadix. Apreliminary report of the results was given earlier (23).

MATERIALS AND METHODSFlowers of Philodendron grandifolium and Peltan-

dra virginica were obtained from the greenhouse atSmith College, Northampton, Massachusetts, and fromlocal swamps, respectively. For measurements of res-piration, sections 0.25 to 1 mm thick were sliced fromthe male portion of the spadix. Oxygen exchange atatmospheric pressure was measured either by conven-tional manometry, with slices suspended in buffer, orby volumetry (21) for slices in a moist gas phase.Oxygen exchange at 7.8 atmospheres was also meas-ured volumetrically. A clear cellulose acetate com-pensating chamber encased an experimental and ablank volumeter of equal dimensions (21). HCN wassupplied from Ca(CN)2-Ca(OH)2 mixtures as de-scribed by Robbie (19). Gas mixtures were made upfrom commercial tanks of 02, N2, and CO (scrubbedwith alkali).

To obtain the particulate preparation, male flow-ers were ground in a small volume of 0.05 M phos-phate buffer, pH 7, which was also 0.3 M with respectto mannitol. The homogenate was transferred to acentrifuge tube and spun at 1,000 x g for 5 minutes.The supernatant was then centrifuged at 10,000 x gfor 15 minutes, and the residue suspended in 1 ml of

1 Received September 21, 1956.2 This work was supported in part by grants from the

American Cancer Society to Dr. K. V. Thimann and toHarvard University.

3 Present address: Department of Botany, CornellUniversity, Ithaca, New York.

4 Present address: Department of Bio'ogy, Universityof Buffalo, Buffalo, New York.

the original grinding medium to give the final par-ticulate preparation. All of these operations werecarried out at 0° C. Cytochrome c oxidase activitywas assayed in a Beckman DU spectrophotometer byfollowingf the decrease in optical density (OD) at 550mm. Cytochrome c obtained from Sigma was re-duced with Na2S204 and the excess hydrosulfite re-moved by aeration.

Difference spectra were computed from the lighttransmission through about 1 cm (= 1.5 Peltandraspadix diameters) of tissue in a moist gas phase. ADC single beam spectrophotometer was operatedmanually as follows: wavelength band-width 3.3 mA,"noise" about 103 OD units, drift 10-3 OD units perminute, galvanometer period 15 seconds. The lightsource was a 6-volt 18-amp ribbon filament lamp fedfrom a storage battery, which was charged at the rateof discharge. The monochromator was a Bausch andLomb 500-mm grating type with 600 grooves/mm. Aphotomultiplier tube (RCA 6217), operated at 700volts, received the transmitted light. The voltagedrop across a 110-megohm load resistor was convertedto galvanometer deflections through a cathode followerbridge (6). Light minus dark deflections were firstrecorded at a series of wavelengths for the tissue inN2. After flushing with 02, the series was repeatedin the same sequence. A similar sequence of meas-urements was then made on the same.tissue in thepresence of HCN.

RESULTSRESPIRATORY STUDIES: Table I shows that 104 M

and 103 M HCN stimulate the respiration of Philo-dendron slices maintained in a moist gas phase. Simi-lar experiments show (table II) that this tissue alsorespires more rapidly in a gas mixture of 95: 5 CO: 02than in 95: 5 N2: 02. From the data of table II it isevident that the respiratory rate in 5 % 02 nearlyequals that in air (21 % 02). This is in marked con-trast to the observation on Arum maculatum (8),where approximately 75 % inhibition of the observed

TABLE ICYANIDE PROMOTION AND INHIBITION OF PHILODENDRON

SPADIX RESPIRATION IN VOLUMETRICRESPIROMETERS

RESPIRATORY MEASUREMENTSHCN

A B

x 10-4M tl 02 x gm fresh wt- x hr' at 250 C0 1320 9701 1620 1420

10 1700 154040 1160 ....

186

www.plantphysiol.orgon January 8, 2019 - Published by Downloaded from Copyright © 1957 American Society of Plant Biologists. All rights reserved.

http://www.plantphysiol.org

YOCUM AND HACKETT-CYTOCHROMES IN AROIDS

TABLE IICARBON MONOXIDE (CO) PROMOrION OF PHILODENDRON

SPADIX RESPIRATION IN VOLUMETRICRESPIROMETERS

AIRN2: 02 CO: 02AIR 95:5 95:5

Al 02 x gm fresh wt-1 x hr-' at 250 C1280 1060 14901710 1380 17301970 1540 2040

rate resulted from reduction of the oxygen partialpressure from 21 to 5 %.

Since both the plant material and the methodsdiffered in the two cases, the dependence of the re-spiratory rate of Philodendron slices on oxygen pres-sure (PO2) was determined by the two methods andthe results compared. Slices respiring in a liquidphase in a Warburg vessel, under conditions approxi-mating those of James and Beevers, show a markeddependence upon P02, as shown in figure 1 (curve B);0.15 atm 02 supports half the maximal rate. On theother hand, if no external liquid is in contact withthe slices the rate of respiration depends upon the pO2only at very low pressures (fig 1, curve A). Underthese conditions, the P0250 (02 partial pressure re-|sSAS;tn1; n n;n1 -Q;t+tr l+ni ;o n M9quireu

atm 0

sentialtissuesatura

1000

0.

a

500

0

0.20

44z

)-. 0.10

z o.osI 0.07A

.90.074' 0000F 0.05

0

a0.03

z10.02

It0a .0

0 I 3- 4 5 6 0

TIME MINUTES2 3 4 5

FIG. 2. Cytochrome c oxidase activity of a particu-late preparation from Peltandra spadix. Final concen-

trations in cuvette: mannitol, 0.1 M, phosphate buffer,0.05 M, pH 7, cytochrome c, 2 x 10' M. The reactionwas initiated by introducing 0.25 ml of the particulatesuspension prepared from 0.050 gm of spadix. The totalvolume was 3.25 ml and the temperature 250 C.

11(ir ['all tIne miaximlaL respirlalUoy lale) is u.wuJ Experiments of the type illustrated in figure 1,

12 Thus, the removal of the external liquid (es- curve A, were also carried out to test the effect of CO

lly water in this case) from the respiring spadix on the oxygen affinity. The results, shown in table

decreases the oxygen pressure required for half III, demonstrate that the affinity is still high; e.g.,

tion about 100 fold. P0250 is attained at 0.004 atm by Philodendron at

250 C. This value may be compared to the P0250 of

A0.002 atm measured in the absence of CO (fig 1).

,,__-- CYTOCHROME C OXIDASE: Peltandra spadix par->.,,- ticles (10,000 x g x 15 min fraction) contain an active

B _-° cytochrome c oxidase as shown in figure 2. First or-I _-der kinetics are followed approximately; a velocity! , constant, k, of 0.36 to 0.38 x min-' was estimated for

pots .002 atm this preparation. The initial rate of cytochrome oxi-

F$,So' dation (k x initial concentration of 2 x 10 5 M) corre-

sponds to an 02 uptake of 2.42 ,ul x hr-1 x ml-'. This/ Po2r@ .16 atm. times the dilution (0.015-1) gives 161 p.1 x hr1 x grams-

for the computed respiratory rate of the spadix whichcan proceed via recovered particulate cytochrome c

0' oxidase. Assuming that approximately 10 % of theo AAIt__' activity of this enzyme is recovered during prepara-

0 .10 .20 .30 .40 .SO LO tion, the computed rate of the spadix respiration be-pop (atmospheres)

FIG. 1. Respiratory rate Qo2 in ul 02 X gm fresh wt-'x hr-1 at 250 C of Philodendron grandifolium spadix slices

as a function of the oxygen partial pressure, PO2. The

data of curve B were determined manometrically in

15-ml vessels shaken at 120 oscillations (4 cm) per min-

ute; 0.4 gm of tissue was suspended in 3.0 ml of 0.06 M

phosphate buffer, pH 7. The data of curve A were de-

termined volumetrically in 4-ml vessels containing 0.2

gm tissue in contact with no external liquid phase. All

data were from the same spadix.

TABLE IIITHE PARTIAL PRESSURE OF 02 REQUIRED FOR HALF MAXI-MAL RESPIRATORY RATE PO25') OF AROID SPADIX TISSUES

RESPIRING IN CARBON MONOXIDE (CO)

PLANT TEMP, 0 C PCO,* ATM PO25, ATM

Philodendron 25 0.996 0.004Peltandra 15 7.8 0.012Peltandra 0 0.998 0.002

* pCO = partial pressure of CO.

o 13z10'4M H1CMo a a03-A

A

A

xNnoH4CN Oa

Ixio- M"CHM Air

187



PLANT PHYSIOLOGY

comes 1600 ,ul x hr-1 x grams-1. The measured spadixrespiratory rate is of this order of magnitude.

Also shown in figure 2 are experiments with inhibi-tors. 1 x 10- M HCN suppressed the rate of cyto-chrome c oxidation by more than one half. A gas

mixture of 0.90 atm CO and 0.10 atm 02 decreasedthe rate constant to 23 % of that in 0.90 atm N2 and0.10 atm 02-

SPECTROPHOTOMETRY: A tissue difference spectrum,i.e., optical density in the reduced state minus that inthe steady state oxidized, is shown in figure 3, curve A.Absorption maxima occur at approximately 605 and550 m,u, which correspond to the a-bands of reducedcytochromes a + a3 and c, respectively. The 550 m,umaximum is larger by a factor of about 2 and showsasymmetry on the long wavelength side, probably dueto absorption by the a-band of a cytochrome b. Theseresults, which show the reduction of these componentsin N2 and their oxidation (at least partial) in 02,

strongly suggest the participation of cytochromes inthe respiration of this tissue.

The difference spectrum was also determined in a

concentration of HCN which does not inhibit respira-tion (see table I) yet inhibits isolated cytochromeoxidase by about 95 %. As shown in figure 3, curve

B, the absorption maxima at 550 and 605 m,u are de-creased by about one half and another maximum at560 m,u (here assumed to be cytochrome b) is evi-dent. There is a suggestion of another absorptionband at 585 to 590 mjA. It is evident that respirationin the presence of cyanide is mediated, at least inpart, by cytochrome components. However, the rela-tive contributions of the individual cytochromes, as

revealed by the difference spectra, are markedly al-tered by cyanide.

+0.0lr

O / \ r~~~~~~oerobie t HCN0

E | "e\.-ano @robic + HON

-0.02 b

0

520 540 560 580 600 620

Wavelength (mpr)

FIG. 3. Difference spectra (reduced minus steadystate oxidized) for the spadix of Peltandra virginwca.Each point for the oxidized state was obtained about5 min after the corresponding one for the reduced state.HCN (2 x 10' M) was furnished by KCN-KOH buffers(19) in the gas train and on filter paper in the light ab-

sorption cell.

TABLE IVOPTICAL DENSITIES, CONCENTRATIONS AND TURNOVER

NUMBERS OF CYTOCHROMES IN A RESPIRINGPELTANDRA SPADIX

CONC OF CYTOCHROMESMEASUREMENTS HCN, M CY bO a+ME

OD (cm-') 0 0.031 0.020 0.012Conc (,uM) 0 1.7 1.1 0.7TN (seC-') 0 30 46 73OD (Cm-') 2 x 10' 0.012 0.020 0.007

The optical density (OD) data were obtained fromthe difference spectra of figure 2 by subtracting the ODat a neutral reference wavelength from that at the ab-sorption maximum: cytochrome c (550 to 540 mu), b (560to 580 m,) and a+ a3 (605 to 615 m,u). An extinctioncoefficient of 18 x mM-' x cm-' was used for each compo-nent (2). For the turnover (TN) estimation, a respira-tory rate of 1000 IAl 02 X gm fresh wt- x hr-1 was assumed.

Of the 6 or so difference spectra obtained by thismethod, 4 showed the 605 m,u peak below the baseline. The pair of curves shown on figure 3 were theonly two that were obtained on the same spadix andthat included the cytochrome a region. The coinci-dence of the two curves (no corrections were appliedto either curve) is evidence at least of their repro-ducibility within a single spadix. Also when severalof the wavelength points were remeasured at the endof a run, they could be reproduced within 103 ODunits: that is the spectrophotometer did not drift ex-cessively.

The concentrations of participating cytochromes,estimated by Chance's method (2) from the differ-ence spectra of figure 3, are shown in table IV. Incontrast to the cytochrome a + a3 and c values, whichare decreased by HCN, the cytochrome b value showedno detectable change. That the steady state oxidizedlevel of cytochrome b is not diminished by HCNpoints to its functioning in respiration both in thepresence and absence of the inhibitor.

DISCUSSIONThe studies reported here show that measured re-

spiratory rates at low oxygen pressures are stronglydependent upon the method of oxygen supply to theAroid spadix. When furnished from a moist gas phase,a high O2 affinity is observed. If, however, a waterlayer separates this rapidly respiring tissue from thegas phase, the apparent affinity is lower by a factorof about 100. We attribute such differences to thefact that the diffusion coefficient of oxygen in wateris 3 x 10h5 times its value in air at 200 C (9). Fromthis, it follows that diffusion through the liquid sus-pending medium also limited the rapid rate of 02 ex-change in the earlier experiments with Arumn macu-latum (8). That this is indeed so has now been con-firmed by Simon's (personal communication) demon-stration of a high oxygen affinity for Arum macula-tum slices respiring in a moist gas phase.

188




Several types of evidence for the participation ofcvtochromes in the respiration of the spadix have beenprovided here. First, a comparison of the oxygenaffinity (1/0250) of the intact tissue with the valuesfor a variety of isolated oxidases (table V) revealsthat it exceeds the affinity of all except cytochromeoxidase. This fact alone argues strongly against thefunctioning of copper- or flavin-containing oxidases inrespiration. The oxygen affinity for the spadix (3 x105) equals that of freshly cut potato tuber slices, inwhich at least 70 % of the respiration is mediated bycvtochrome oxidase (21). On the other hand, thevalues for yeast (1 x 106) and Aerobacter (3 x07),both of which utilize a cytochrome system, are higher.We attribute this difference to longer oxygen diffusionpaths within the tissues than within the microorgan-isms.

The second type of evidence is the demonstrationof cytochrome c oxidase in extracts of Philodendronand Peltandra. Both with respect to its inhibitorsensitivity (fig 2) and its intracellular localization (onparticles), the spadix enzyme is similar to the cyto-chrome oxidase isolated from other cells. This activityis generally ascribed to cytochromes a + a3, suggestingthat these components are present. The enzyme isprobably present in all spadices, since it has also beendemonstrated in Symplocarpus foetidus (1, 7). How-ever, this evidence alone does not prove the partici-pation of cytochrome oxidase in the respiration ofintact tissues.

Further evidence has been supplied by spectro-photometric observations on intact tissues. The dif-ference spectrum (fig 3) shows that cytochromes band c, as well as cytochrome oxidase, are involved inthe reaction with molecular oxygen. The concentra-

TABLE VOXYGEN AFFINITIES (1/K6, OR 1/[02]5) * FOR RESPIRA-

TION AND OXIDASES

TEMP, [O]5 1/[t02] REF0c ~~ORl1/Km, E

Respiration1. Aerobacter 19 3.1 x 10' 3.2 x 10' (12)2. Yeast 20 1 x 10' 1 x 106 (22)3. Potato (tuber slices) 25 3 x 10' 3 x 10 (21)4. Aroid spadix 25 3 x 10-' 3 x 10W (fig 1)

Oxidases1. Cytochrome c 20 2.4 x 10' 4 x 10' (12)2. Ascorbic acid 30 3 x 10' 3 x 103 (15)3. Ascorbic acid 25 1.5 x 10' 6 x 10' (21)4. Phenol 25 3x 10-5 3x104 (10)5. Amino acid 38 5x10-' 2x104 (11)6. Xanthine 38 7 x 10' 1.4 x 104 (11)7. Glucose (notatin) 38 1 x 10i 1 x 104 (11)

[O2]°' is the oxygen concentration, in moles per liter,required for half the maximum rate of oxygen uptake.

p02' X C 02[O2,Y =

22.4 liter atm/molewhere: pO2, is the partial pressure, in atmospheres, ofoxygen required for half the maximum rate of oxygenuptake. °CO2 is the oxygen solubility coefficient.

tions of the cytochromes functioning in respiration, ascomputed from the optical density differences (fig 2)and the extinction coefficients are included in tableIV. Both the absolute (,- 10-6 I) and relativeamounts are similar to those reported for other aero-bic cells (2, 13). From these data and the spadix re-spiratory rate, turnover numbers were estimated forthe cytochromes a + a3, b, and c. The turnover num-bers are within the range of published values of 10 to100/ sec for yeast, muscle and roots (2, 13). Thus arespiratory chain of the b -- c -- a + a3 type can ac-count for the spadix respiration. In spite of this evi-dence that the Aroid spadix contains and uses a com-plete cytochrome system, the tissue respiration showsa remarkable insensitivity to cytochrome oxidase in-hibitors, and this observation remains to be explained.

Several possible causes of this insensitivity to in-hibitors are considered: 1) The oxidation of CO toCO, with a consequent uptake of oxygen, could maska respiratory inhibition. This possibility has beeneliminated for CO-insensitive potato slices (21) andmature leaves (4) and it is not suggested for theAroid spadix, since the volume of gas consumed neverexceeded the initial volume of oxvgen. 2) The oxy-gen-activating system might be protected in somemanner from the inhibitors. Evidence that CO andcyanide do in fact combine with some respiratorycomponent is provided by the observed stimulation ofrespiration (tables 1,1) and by the alteration in thedifference spectrum induced by cyanide (fig 3). 3)The presence of a large excess of cytochrome oxidaserelative to the rate limiting step in respiration couldaccount for the tissue's insensitivity to HCN and CO.This hypothesis has been advanced recently to ac-count for the case of mature leaves (5). The quan-tity that would be required for the Aroid spadix re-spiring in CO may be computed by assuming that (a)all electrons are transferred to cytochrome c oxidase(cytochrome a3) and (b) the following modificationof Warburg's partition equation describes the CO in-hibition of respiration:

n (CO) =K(a3) - n (02)

wheren= residual quantity of the oxidase, relative

to an amount 1 required for the respira-tory rate in air.

(a3) = total quantity of the oxidase, relative toan amount 1 required for the respiratoryrate in air.

(CO) = CO partial pressure.(02) = 02 partial pressure.K= relative affinity constant of the oxidase

for 02 and CO.

K was computed from 77 % inhibition of Peltan-dra cytochrome c oxidase preparation by a 9/1 ratioof C 0110 (fig 2) and found to be 2.7. This is in closeagreement with Winzler's value of 2.5 from yeast res-piration experiments (22).

189



PLANT PHYSIOLOGY

From the CO/02 ratio (table III) required to in-hibit the respiratory rate by 50 %, i.e., when n = 0.5,(a3) was found to lie between 47 and 129 (,- 100).The oxidase turnover number resulting from - 100fold excess over the rate limiting step would be

100-1 that in cells where there is evidence for nolarge excess (yeast, e.g., 22). However, the meas-ured spadix oxidase turnover number of 73 (sec-1) isin close agreement with that of yeast (2). This sug-gests an activity not large enough, in relation to therate limiting step, to support the excess hypothesis.4) Another possibility is that this tissue possesses analternate, cyanide-resistant pathway of electron trans-fer to oxygen. Such a proposal was first made byOkunuki (17) for pollen, as early as 1939. Positiveevidence that this is the case in the Aroid spadix canbe obtained from the spectrophotometric observations(fig 3). The rate of oxidation of cytochrome b, incontrast to c and a + a3, is not suppressed by 2 x 104 MHCN. This finding is consistent with the followingscheme of electron transport pathways:

Su bstrate-. b

HC

- 02-0 X - b°

We have assumed a single cytochrome b and a seriestransfer of electrons among the components whereverpossible. In the presence of inhibitor, some of theelectrons proceed via an alternate pathway, which in-cludes cytochrome b and possibly another oxidase,designated here as x. The remainder of the electronsare transferred to oxygen via the partially inhibitedc -- a + a3 02 system. The rates of transfer throughthe two systems will depend on the amount of enzymenot combined with inhibitor and the rate of reductionof the oxidases. The possibility that an inhibitor al-ters the system which it is being used to study sug-gests a situation of experimental "indeterminacy."

Although the nature of component x in the abovesequence is not known, some of the properties can bededuced. This component, which has little or no affin-ity for HCN and CO, shows a high affinity for oxygen(1/[02150 in CO = 1.5 x 105 M) and is able to mediatea relatively rapid rate of oxidation. The simplestpossibility would be that cytochrome b is oxidized di-rectly by oxygen; the cytochromes of the b group are,in fact, autoxidizable substances which do not com-bine with cyanide or CO. The spectrophotometricmethod employed here does not exclude the possibilityof other functional cytochromes of the b class. How-ever, low temperature (-196° C) spectroscopy has re-

solved only one component with a band absorbing inthe wavelength region of cytochromes b (1). In a

study carried out independently from the presentwork, Lundegardh (13) demonstrated spectrophoto-metrically that the cytochrome b of roots remains par-tially oxidized in aerated cyanide, and he has sug-gested that "the autoxidation of cytochrome b could

possibly act as a substitute for cytochrome oxidase inthe living cell." Nevertheless, the rapidity of the oxi-dation, turnover number = 45/sec, suggests that an ad-ditional component, above the potential of cytochromeb, functions as an oxidase in this system. By analogywith the classical system, one might expect that amember of the cytochrome a family, with a low affin-ity for CO and HCN, might serve as the terminal oxi-dase. The presence of an absorption banld of 590 muin the difference spectrum in cyanide suggests the pos-sible functioning of an a-type cytochrome. A band atapproximately the same wavelength is also observedat - 1960 C in reduced Symplocarpus particles in theabsence of HCN (1).

The cytochromes b5 and b3 have been proposed asmembers of cyanide-resistant respiratory pathways inanimal (18) and plant (16) cells respectively. Butthese hypotheses are not supported by the followingevidence. The turnover number for cytochrome b-of 0.13/sec. (3) and an estimate computed from thedata of Martin and Morton (16) of 0.05 for cyto-chrome b3 are insufficient, being > 300Y times the45/sec computed here for cytochrome b of the Aroidspadix. In addition, cytochromes b3 and b5 are local-ized on microsomes, particles of too low oxidase ac-tivity to account for the respiration of the tissue fromwhich they were isolated (7, 20).

It is of considerable interest to know whether elec-tron transfer through an alternate pathway is coupledto oxidative phosphorylation. The increase in res-piration caused by CO and cyanide could be the re-sult of an "uncoupling" of phosphorylation from thenormal respiration, and the activation of a non-phos-phorylating electron transport system; oxidation viathe latter pathway would not be limited by the sup-ply of phosphate acceptors.

A cyanide and/or CO-insensitive respiration hasbeen demonstrated with a wide variety of plants, ani-mals, and microorganisms. In many, although not all,of these cases it has also been shown that the cells con-tain cytochrome components, including cytochromeoxidase. On the basis of our own experiments, it issuggested that the "normal" respiration of such cellsmay be mediated by a classical-type cytochrome sys-tem and that an alternate pathway may be activatedin the presence of oxidase inhibitors, such as HCNand CO.

SUMMARYThe following evidence is presented for the par-

ticipation of cytochromes in the respiration of theAroid spadix:

(a) The spadix respires with a high affinity foroxygen, provided the rate of oxygen supply isadequate.

(b) A particle-bound cytochrome oxidase withhigh affinity for cyanide and carbon monoxidecan be demonstrated.

(c) Different spectra of the intact spadix show ab-sorption maxima at wavelengths correspond-ing to the a-bands of cytochromes a+a3, b

190




and c. The molar concentration of each is ofthe order of 106.

(d) Turnover numbers of 10 to 102/sec, estimatedfrom cytochrome concentration and respira-tory rate, fall within the published values forcytochromes from yeast, muscle, and roots.

The cyanide and carbon monoxide-resistant res-piration of the Aroid spadix can be accounted for bythe oxidation of a cytochrome b, either directly byoxygen or via an oxidase without appreciable affinityfor these inhibitors.

Since the completion of this work, the paper ofD. S. Bendall and R. Hill, on "Cytochrome compo-nents in the spadix of Arum maculatum" (New Phy-tologist 55: 206-212. 1956) has come to our atten-tion. In addition to cytochromes a, b, and c, the par-ticulate fraction was shown to contain a componentcytochrome b7, which is rapidly oxidized even in10-3 M cyanide.

We are indebted to Dr. K. V. Thimann for en-couragement throughout the course of this work, andto Dr. W. D. Bo.nner, Jr., for the use of a spectro-photometer and a refrigerated centrifuge.

LITERATURE CITED1. BONNER, W. D. and YOCUM, C. S. Spectroscopic and

enzymatic observations on the spadix of skunkcabbage. Plant Physiol. 31 Suppl.: xli. 1956.

2. CHANCE, B. Spectra and reaction kinetics of re-spiratory pigments of homogenized and intactcells. Nature 169: 215-221. 1952.

3. CHANCE, B. and PAPPENHEIMER, A. M., JR. Kineticsand spectrophotometric studies of cytochrome b.,in midgut homogenates of Cecropia. Jour. Biol.Chem. 209: 931-943. 1954.

4. DALY, J. M. Stimulation of respiration by carbonmonoxide. Arch. Biochem. Biophys. 51: 24-29.1954.

5. DUCET, G. and ROSENBERG, A. J. Activites respira-toires chez les veigetaux superieures. Giorn. bio-chim. ital-franco-elvetiche 25: 214-218. 1955.

6. GLASSER, L. G. and TROY, D. J. A new high sensi-tivity differential colorimeter. Jour. Optical. Soc.Amer. 42: 652-660. 1952.

7. HACKETT, D. P. Respiratory mechanisms in theflowers of skunk cabbage. Plant Physiol. 31 Suppl.:xli. 1956.

8. JAMES, W. 0. and BEEVERS, H. The respiration ofArum spadix. A rapid respiration resistant tocyanide. New Phytologist 49: 353-374. 1950.

9. KROGH, A. The Comparative Physiology of Re-spiratory Mechanisms. Pp. 1-172. Univ. of Penn-sylvania Press, Philadelphia 1941.

10. KUBOWITZ, F. Spaltung und Resynthese der Poly-phenoloxydase und des Hiimocyanins. Biochem.Zeits. 299: 32-57. 1939.

11. LASER, H. The effect of low oxygen tension on theactivity of aerobic dehydrogenases. Proc. Roy.Soc. (London) B 140: 230-243. 1952.

12. LONGMUIR, I. S. Respiratory rate of bacteria as afunction of oxygen concentration. Biochem. Jour.57: 81-87. 1954.

13. LUNDEG'ARDH, H. Properties of the cytochrome sys-tem of wheat roots. Physiol. Plantarum 5: 97-146. 1952.

14. LUNDEGARDH, H. On partial oxidation of the cyto-chlome system in the presence of cyanide. Physiol.Plantarum 8: 95-105. 1955.

15. MANDELS, G. R. The atypical ascorbic acid oxidasein fungus spores. Its inactivation by isoascorbateand its specificity. Arch. Biochem. Biophys. 44:362-377. 1953.

16. MARTIN, E. M. and MORTON, R. K. Cytochrome bsof microsomes from plant tissues. Nature 176:113-114. 1955.

17. OKUNUKI, K. tVber den Gaswechsel der Pollen, II.Acta Phytochim. (Japan) 11: 27-64. 1939.

18. PAPPENHEIMER, A. M. and WILLIAMS, C. M. Cyto-chrome b5 and the dihydrocoenzyme I-oxidasesystem in the Cecropia silkworm. Jour. Biol.Chem. 209: 915-929. 1954.

19. ROBBIE, W. A. The quantitative control of cyanidein manometric experimentation. Jour. CellularComp. Physiol. 27: 181-209. 1946.

20. STRITTMATTER, C. F. and BALL, E. G. The intracel-lular distribution of cytochrome components andof oxidative enzyme activity in rat liver. Jour.Cellular Comp. Physiol. 43: 57-78. 1954.

21. THIMANN, K. V., YocuM, C. S. and HACKETT, D. P.Terminal oxidases and growth in plant tissues.III. Terminal oxidation in potato tuber tissue.Arch. Biochem. Biophys. 53: 239-257. 1954.

22. WINSLER, R. J. A comparative study of the effectsof cyanide, azide, and carbon monoxide on therespiration of bakers' yeast. Jour. Cellular Comp.Physiol. 21: 229-252. 1943.

23. YocuM, C. S. and HACKETT, D. P. Terminal oxida-tion in the Aroid spadix. Plant Physiol. 30 Suppl.:xxx. 1955.

191



Documents

Smith College, Northampton, Massachusetts, and - Plant Physiology