Structure of the Respiratory Chain System as Indicated Studies with Hemophilus parainjluenzae*

Transcription

1 TH JOURNAL OF B~omorca~ CHUI~TRY Vol. 237, No. 4, April 1962 Printe in U.S.A Structure of the Respiratory Chain System as Inicate Stuies with Hemophilus parainjluenzae* by LUCIL ShmHt AND DAVID C. WHITS From the Department of Biochemistry, Dartmouth Meical School, Hanover, New Hampshire (Receive for publication, July 18, 1961) Stuies on the respiration an the cytochrome system of Hemophilus parainsuenzae have been escribe in the preceing paper (1). Some of the unusual properties of the respiratory chain system of these organisms have affore a new experimental approach to the nature of this multienzyme system. H. parainfiuenzae harveste in the stationary growth phase contains a number of ifferent cytochromes, incluing a relatively large quantity of a cl-type cytochrome. The extent of reuction of the latter, when the bacteria become anaerobic, is ifferent in the presence of succinate from what it is in the presence of reuce iphosphopyriine nucleoce. A consierable proportion of this cytochrome is not reuce anaerobically in the presence of either substrate. When the cells are broken, the cytochrome cl which is not reuce enzymatically is foun in solution, an further amounts of the particle-boun cytochrome can be extracte by washing with buffer. The respiratory rate of the particles oes not ecrease until a large amount of the cytochrome cl has been remove. The cytochrome in solution oes not interact with the particulate oxiases an reuctases. From this type of observation, inferences can be rawn concerning the structure of the electron transport system. XPRJMNTAL PROCDUR Growth of Bacteria an of Respiratory Particles- The methos for culturing the bacteria are escribe in the preceing paper (1). Respiratory particles are prepare by grining a paste of washe cells with 3 to 4 times its weight of Alcoa alumina A-301 in a col mortar, then extracting with about 4 volumes of 0.5 M sucrose containing 0.05 M phosphate buffer, ph 7.6. The alumina is remove by centrifugation at 2,000 X g for 15 minutes. Then centrifugation at 6,000 X g for 15 minutes removes most of the unbroken cells, an a further centrifugation at 8,000 X g carries own the remaining whole cells plus some large particles, as shown by examination with the phase contrast microscope. Most of the insoluble particles containing the respiratory chain enzymes, which are suspene in the supernatant flui, can be collecte by centrifugation at 35,000 X g for 30 minutes. All of these particles can be centrifuge own by exposure to 140,000 X g for 30 minutes. The insoluble particles can thus be washe with buffer by centrifu- * This research was supporte by a grant from the Unite States Public Health Service. t Senior Fellow, Unite States Public Health Service. $ Postoctoral Grauate Fellow, Rockefeller Institute, New York, New York. gation an resuspension in buffer with the ai of a Teflon homogenizer. Respiration Measurements-Respiration of the bacteria or respiratory particles with ae DPNH or other substrates was measure with the platinum oxygen electroe, as escribe in the preceing paper (1). Oxygen uptake of the particles with silicomolybate as the electron onor was followe similarly, using the reagent escribe by Jacobs (2) in the presence of 1 mm ascorbate, ph 7.0. Difference Spectra-The ifference in absorption spectrum between anaerobic an aerobic suspensions of intact bacteria was measure either in the split-beam recoring spectrophoto- meter in the Johnson Founation of the University of Pennsylvania, through the courtesy of Dr. Britton Chance, or in the Gary moel 14 recoring spectrophotometer. When ifference spectra of intact bacteria were measure in the Cary spectrophotometer, the bacteria were suspene in 50% glycerol by volume. Difference spectra of broken-cell suspensions or of washe particles in buffer were also measure in the Cary spectrophotometer. Details of the proceures are given in the preceing paper (1). The plotting error in the Cary recorings amounte to in optical ensity, using the 0 to 0.1 slie wire; the absorption spectra have been trace with a line through the mile of the tracing error. Reuction of Ferricyanie-The rate of reuction of ferricyanie was assaye by measuring the change in optical ensity at 429 rnp at 25 with a reaction mixture containing 0.05 M phosphate buffer (ph 7.6), 0.5 mm potassium ferricyanie (freshly prepare), 5 mm potassium cyanie, 0.02 M soium succinate or other acis teste as substrates or 5 mm DPNH, an bacterial particles containing 0.5 to 1 mg of protein in a final volume of 3 ml. Protein Determination-The protein content of the bacterial or particle suspensions was measure by the Gornall biuret metho (3) in the presence of 0.06% soium eoxycholate. Reagents-All chemicals were reagent grae commercial proucts, use as obtaine, except for soium succinate, which was recrystallize twice from hot mater by the aition of alcohol. RSULTS Difference spectra of Intact Cells an Cell-free xtracts-the otte line of Fig. 1 plots the ifference in absorption spectrum between an aerobic suspension of washe H. parainjluenzae an some of the same suspension which has exhauste the oxygen in solution on oxiizing succinate (the epletion of oxygen 1337

2 1338 Respiratory Chain System of H. parainjiuenxae Vol. 237, No o.a i!! 0 C 6 xi toi i I I I I I I I I W ovelength FIG. 1. Difference spectra of intact cells. The suspension of washe bacteria in buffer containe 7.4 mg of protein per ml. In each case the reference cuvette containe aerobic bacteria with no ae substrate. For the otte cutz)e, succinate was ae to the other cuvette an the suspension allowe to become anaerobic. Then DPNH was ae an the ashe curse recore after 9 minutes, when no further change in optical ensity took place. After the aition of a few grains of Na&Oa, the soli curve was trace. The ifference spectra were measure in the split-beam recoring spectrophotometer in the Johnson Research Founation. being measure by the oxygen electroe). As explaine in the preceing paper, a-absorption peaks corresponing to cytochrome u2, al, an cl are reaily istinguishe at 630, 600, an 553 mp, respectively. When DPNH is then ae to the anaerobic cells containing succinate, the peak in the absorption spectrum at 553 rnp increases until it reaches the value seen in the ashe curve. Then aition of Na2Sz04 immeiately results in further reuction of the cytochrome cl (soli curve). The extent of reuction of cytochromes al an a2 is essentially the same in all three conitions. Fig. 2 plots similar ifference spectra of a culture having higher levels of cytochromes al an u2 an shows more clearly that cytochromes al an u2 are completely reuce in anaerobic cells containing DPNH. The amount of cytochrome cl reuce anaerobically with DPNH is 5 to 10 times greater than the amount of cytochromes al, ~2, b, or o. The excess of cytochrome c1 which can be reuce only with Na2S204 is variable in ifferent cultures an particularly seems to vary with the age of the culture. The same extent of reuction of cytochrome cl is observe in anaerobic cells containing DPNH whether succinate is present or not; that is, the extent of reuction of the cytochrome cl by succinate an DPNH is not aitive. The aition of 1 mm cyanie oes not increase the extent of reuction of cytochrome c1 by succinate or DPNH. (mi The wave lengths of the absorption peaks in the anaerobic minus aerobic ifference spectra of broken-cell suspensions or washe particles of H. puruinfluenzue are the same as those obtaine with intact bacteria. An in the cell-free extracts the increase in the reuction of the cytochrome cl on aition of DPNH to cells anaerobic in the presence of succinate takes place immeiately. Fig. 3 plots the ifference spectra of particles washe once, from which 53% of the cytochrome cl of the cell-free extract has been remove (see below). The anaerobic minus aerobic ifference spectrum shows evience for an absorption peak corresponing to a b-type cytochrome (absorption peak at 560 mp). This, plus the absorption spectra of bacteria inhibite with 2-n-heptyl-4-hyroxyquinoline-N-oxie (1) form the evience for the participation of a b-type cytochrome in the respiratory chain of these bacteria. In the presence of the large quantity of cytochrome cl usually present, the absorption peak of cytochrome b is maske. The carbon monoxie ifference spectrum of the particles shows absorption peaks corresponing to cytochromes o an al. The respiratory particles can only respire in the presence of succinate or DPNH, an the rate of respiration is aroun 6- to 20.fol more rapi with DPNH than with succinate. The characteristics of the respiratory particles will be escribe in a separate publication. Separation of Soluble Cytochrome cl from Cell-free xtracts an Removal of Cytochrome from Respiratory Particles---When a freshly prepare broken-cell extract is centrifuge for 45 minutes at 140,000 x g, some of the cytochrome cl remains in the * k al O Wavelength FIG. 2. Same as in Fig. 1; the soli curve was obtaine with anaerobic cells in the presence of DPNH, then the ashe curve was recore after aition of a small quantity of soli NazSz04. The absorption spectra were measure in the Cary moel 14 spectrophotometer with a strong suspension of bacteria (14.7 mg of protein per ml) in 50$& (volume per volume) glycerol. (mp)

3 April 1962 L. Smith an D. C. White 1339 supernatant flui. The remainer of the cytochrome c1 an all of the other cytochromes are centrifuge own with the insoluble pellet. The ata of Table I show that the cytochrome cr in the supernatant flui is exactly equal to the amount of this cytochrome in the whole extract which cannot be reuce enzymatically in the presence of DPNH, but can only be reuce by Na&04. In other wors, only the particle-boun cytochrome can be reuce enzymatically. Aitional cytochrome cl is progressively remove from the particles by washing with buffer by centrifugation an suspension; in each case only the cytochrome remaining on the particles can be reuce enzymatically (Table I). The supernatant flui from centrifugation of the broken-cell extract an the washings of the particles show absorption spectra typical of cytochrome cl; these plus the absorption spectrum of the pyriine hemochromogen are shown in the preceing paper (l), Fig. 5. The cytochrome cl can amount to 5 to 6% of the protein of the bacteria, assuming that the molecular weight an extinction coefficients of the cytochrome are similar to those reporte for mammalian cytochrome cl. After consierable cytochrome cl is remove from the particles by washing, the respiration rate begins to ecrease, reaching a low value after 3 to 5 washings (Table II), but some ability to respire remains. In one experiment, it was foun that freezing an thawing of the washe particles resulte in a further release of cytochrome cl an a complete loss of the ability to oxiize succinate or DPN H with oxygen. +o.tt % 0 - +o.o + Q 2 Ii Wavelength FIG. 3. Difference spectra of respiratory particles. Dashe line ifference in absorption spectrum between anaerobic particles containing succinate an aerobic particles (no substrate ae). Soli line ifference in absorption spectrum between anaerobic particles in the presence of DPNH an aerobic particles. Dotte line ifference in absorption spectrum between anaerobic particles (plus DPNH) in the presence an absence of carbon monoxie. The perparation was a once-washe particle suspen- sion containing 7.0 mg of protein per ml. The absorption spectra were recore in the Cary spectrophotometer. (mp) TABL I Removal of cytochrome CI from respiratory particles by washing The broken-cell extract containing 4.3 mg of protein per ml was centrifuge at 140,000 X g for 30 minutes, an the pellet resuspene in 0.05 M phosphate buffer, ph 7.6. Subsequent washings were carrie out similarly. The cytochrome cl in the suspensions was assaye by measuring the increase in optical ensity on aition of DPNH, then the further increase on aition of Na&Ol. The cytochrome cl in the supernatant fluis was measure after aition of NazS204. AODSU,,,,, refers to the ifference in optical ensity at this wave length between the oxiize an the reuce forms of the cytochrome. I Rei%J!Iby Centrifuge cell-free ex tract Once washe particles Twice washe particles Cytochrome tt (AODsss m,u) TABL II Removal of cytochrome cl from respiratory particles The broken-cell extract (8.2 mg of protein per ml) was centrifuge at 30,000 X g for 30 minutes; then the pellet was resuspene in 0.05 M phosphate buffer, ph 7.6, to the volume of the original suspension. Subsequent washings were carrie out in the same manner. The cytochrome cl remove by the washings was assaye by measuring the increase in optical ensity in the supernatant fluis on aition of Na&O1. Broken-cell extract Particles washe once. Particles washe 2 times. Particles washe 3 times. Particles washe 4 times. Cytochrome t, remove, AODssa mp Respiration, JSM 0% se0 + Succinate + DPNH The composition of the meium in which the particles are suspene seems to make no ifference in the amount of cytochrome cl which can be washe off; phosphate buffer from 0.05 to 0.5 M an various sucrose concentrations were teste. Fig. 3 shows the ifference spectra of particles washe once. After the particles are washe about 3 times, the anaerobic minus aerobic ifference spectra show an aitional absorption peak at 390 rn.c; the nature of the compoun responsible for this absorption peak is not known. Aition of Soluble Cytochrome cl to Washe Particles-The low rate of respiration of the severalfol washe particles is not increase by the aition of the supernatant flui obtaine on centrifugation of the broken-cell extract. The soluble cytochrome cl which was concentrate an slightly purifie by precipitation with neutralize ammonium sulfate, then ialysis against col water, also ha no effect on the respiration of the washe particles. The soluble cytochrome cl was not reuce by the particles in the presence of DPNH an cyanie, although ferricyanie was rapily reuce (see following section). Reuction of Fe&cyanie an Oxiation of Silicomolybute by

4 1340 Respiratory Chain System of H. parain$uenxae Vol. 237, No. 4 TABL Ferricyanie reuction by repeate1 III y washe respiratory particles The washe particles were prepare as escribe in Table I. For each assay, 0.2 ml of particles was ae to make a total volume of 3 ml. Particles washe once... Particles washe 2 times.... Particles washe 3 times.... Particles washe 4 times.... Ferricyanie reuction with DPNH first orer rate constant sx Respiratory Particles--The respiratory particles show the ability to reuce ferricyanie in the presence of succinate or DPNH, but not with acetate, citrate, malate, or glucose. The reaction was foun to be first orer with respect to the concentration of ferricyanie; thus the relative rates are expresse as the first orer rate constants. With a number of ifferent preparations the rate constant with DPNH was between 10 an 18 times greater than that with succinate. Table III summarizes the observations of ferricyanie reuctase activity of a suspension of progressively washe particles. This activity oes not ecrease on washing, an in fact increases slightly. The ability to oxiize silicomolybate also remains associate with the particles after several washings. In the mammalian respiratory chain system, silicomolybate reacts with the cytochrome oxiase part of the chain, an cytochrome c is require for the reaction (4). The ata show that both the oxiase an reuctase ens of the respiratory chain system of H. parainfuenzae remain attache to the particles uring repeate washings. DISCUSSION Some of the unusual properties of the respiratory chain system of H. parainfiuenzue give inications about the structure of this complicate multienzyme system, which is attache in some way to a lipoprotein membrane within the cell 1. There can be a large excess of one cytochrome (cl-type) over the others, yet all of the cytochrome cl that is particle boun can become oxiize an reuce uring electron transport. Once the cytochrome is remove from the particles into solution, it can no longer interact with the particulate oxiases an reuctases; this appears to be true both with intact bacteria an with isolate respiratory particles. Thus the loss of ability to interact oes not result from the extraction of an aitional essential factor from the particles. 2. A consierable proportion of the cytochrome cl can be remove from the respiratory particles before a ecrease in respiration rate is observe. 3. More of the cytochrome cl is reuce in the absence of air when the system is oxiizing DPNH than when succinate is the substrate oxiize, but cytochromes al an u2 are completely reuce uner both conitions. The reuction of the cytochrome cl with DPNH is the same as it is in the presence of both DPNH an succinate. The rate of reuction of ferricyanie by the respiratory particles plus DPNH can be as much as 20 times greater than the rate of reuction of succinate, although the extent of reuction of the cytochrome cl by DPNH in the absence of air is usually only twice that with succinate. 4. Although a large proportion of the cytochrome cl can be remove from the respiratory particles by washing, the other cytochromes remain firmly attache, as o the oxiase an the DPNH ferricyanie reuctase activities. The observations summarize rener untenable the concept of assemblies of respiratory pigments consisting of 1 molecule of each type. The ata can only be explaine in terms of a 3-imensional array in which electrons can be transferre through any number of pigments attache to the structure, so that loss of some of the pigments oes not result in a ecrease in the over-all rate of electron transport. The ata also require that the DPNH reuctase system have more points of attachment to the respiratory chain system than the succinate reuctase system. These reuctases cannot be attache to separate pathways, since the extents to which they reuce cytochrome cl are not aitive. Fig. 4 is a schematic representation of the ieas expresse above. The usual sequence of electron transport from b-type to c-type cytochromes to the oxiases is assume by analogy with other systems stuie (5-7). It is possible that the sequence is maintaine by the spatial arrangement of the pigments. There is some evience that the same sequence is not always maintaine when the insoluble respiratory chain particles are broken own into erivative pieces (8, 9). In the mammalian respiratory chain system, cytochrome c is attache to the structure in a manner that allows its removal from intact mitochonria or from muscle mince, but not from the isolate respiratory particles, by treatment with relatively high concentrations of salts (10, 11). Cytochrome cl can be separate from the mammalian structure only by more rastic treatment, such as mixtures of etergents an salts, with or without organic solvents or igestive enzymes (12-15). A cl-type cytochrome can be remove from Azotobacter vinelunii by shaking with butanol (16). The attachment of the cytochrome cl of H. parainfluenzae to the insoluble structure containing the respiratory chain appears to be ifferent from any of these, since it is remove from the structure simply by suspening the particles in aqueous solutions an the ionic composition of the meium appears to have little effect on this. There seems to be a kin of equilibrium between the particle-boun cytochrome cl an that in solution, since a certain amount will come off of the particles when they are suspene in buffer, then more is remove by centrifuging them own an resuspening in fresh buffer. The cytochrome goes from the particles into solution even at relatively high concentrations of cytochrome cr in solution. In most other bacterial systems that have been investigate, all of the cytochromes are resistant to removal from the insoluble respiratory particles; the cytochromes of H. parainfluenzae other than the cytochrome cr remain firmly boun. DPNH- b - CI - c, FIG. 4. Schematic representation of the structural arrangement of the cytochrome pigments in the respiratory chain system. The letters c, CI, 6, al, a~, an o refertocytochromes, FP toflavoprotein.

5 April 1962 L. Smith an D. C. White 1341 The soluble cytochrome cl of H. parain$uenzae oes not interact rapily with the oxiases or reuctases of the respiratory particles. It seems highly unlikely that in this case the lack of reaction coul be a result of a enaturation of the cytochrome cr. This coul mean that the oxiation-reuction reactions require some spatial arrangement of the pigments within the lipoprotein membrane. Wiely varying observations have been mae of the reactions of isolate cl-type cytochromes from mammalian sources or from other bacteria with particulate oxiases or reuctases. One kin of mammalian preparation was neither oxiize nor reuce by enzymes on heart muscle respiratory particles (14), whereas another preparation was reporte to be enzymatically oxiize an reuce by a heart muscle fraction, but it is not clear how rapi these reactions were (13). Both a c-type an a cl-type cytochrome isolate from A. vinelunii were oxiize an reuce by particulate enzymes from these bacteria, but not by those of other bacteria or of mammalian heart muscle 06). The observations on the cytochrome system of H. parainfluenzae have some implications concerning cytochrome synthesis. It appears that the synthesis of one of the cytochromes continues until there is a large excess of this pigment, then finally continues after the bacteria can incorporate it into the membrane containing the electron transport system. The ata suggest that the cytochrome is synthesize in soluble form, then incorporate into the membrane. Further stuies are in progress on this subject. SUMMARY The s-type cytochrome of stationary phase cells of Hemophilus parainjiuenzue is only partly reuce when the bacteria are anaerobic in the presence of succinate. Further reuction of cytochrome cl is observe on aition of reuce iphosphopyriine nucleotie to the anaerobic suspension, an still further reuction follows aition of NaG%04. Cytochromes al an a2 are completely reuce in all three conitions. When the cells are broken, cytochrome cl is release into solution in amount equal to the portion in the bacteria which can- not be reuce enzymatically. The remainer of the cytochrome cl an the other cytochromes remain boun to insoluble particles. More of the cytochrome cl can be remove from the particles into solution by washing with buffer. The respiration rate of the particles oes not change until consierable cytochrome cl has been remove by washing. The cytochrome cl in solution is neither oxiize nor reuce by the oxiases or reuctases of the respiratory particles. It oes not increase the respiration rate of particles which have been eplete of cytochrome cl by repeate washing. Washing oes not remove the ability of the particles to reuce ferricyanie in the presence of reuce iphosphopyriine nucleotie or succinate or to oxiize silicomolybate. These observations lea to hypotheses about the structure of the insoluble respiratory chain system. RFRNCS 1. WHIT, D. C., AND SMITH, L., J. Biol. Chem., 237, 1332 (1962). 2. JACOBS,.., Biochim. et Biophys. Acta, 22,583 (1956). 3. GORNALL, A. G., BARDAWILL, C. J., AND DAVID, M. M., J. Biol. Chem., 177, 751 (1949). 4. JACOBS,.., AND SANADI, D. R., Biochim. et Biophys. Acta, 38, 12 (1960). 5. KILIN, D., AND SLATR,. C., Brit. Me. Bull., 9, 89 (1953). 6. CHANC, B., AND WILLIAMS, G. R., in F. F. NORD (itor), Avances in enzymology vol. 17, Interscience Publishers, Inc.; New York, 1956, p SMITH, L., T he ba&.&a, Vol. II, Ch. 7, Acaemic Press, Inc., New York, CHANC, B., Nature, 169, 215 (1952). 9. STABROOK, R. W., J. BioZ. Chem., 227, 1093 (1957). 10. Tsou, C. L., Biochem. J., 60, 493 (1952). 11. JACOBS,.., AND SANADI, D. R.. J. BioZ. Chem (196Oj.,, 12. KILIN, D., AND HARTR,. F., Nature, 176,200 (1955). 13. SKUZU, I., 0~11, Y., AND OKUNUKI, K., J. Biochem. (Tokyo), 48, 214 (1960). 14. GRN, D.., JARNFLT, J., AND TISDAL, H. D., Biochim. et BioDhvs. Acta (1959). 15. BRNSTIN,. H.; AND W~INI~, W. W., Arch. Biochem. Biophys., 91, 138 (1960). 16. TISSI&RS, A., AND BURRIS, R. H., Biochim. et Biophys. Acta, 20, 436 (1956).