Pigment Systems..................................... 243Reaction Centers..................................... 244Quinones as Electron Acceptors..................................... 245Ferredoxin..................................... 247Cytochrome c as Donor for Reaction Center Bchl ................................ 248Primary Photochemical Act .............. ....................... 249
SECONDARY BIoCHEMicAL REACTIONS.................................... 250NAD Photoreduction ..................................... 251Photophosphorylation and Energy Transfer..................................... 253
INTRODUCTIONThe past few years have been active and re-
warding ones for those studying photosynthesisin bacteria, especially in the areas of the earlyphotochemical events and related electron transferand energy transfer reactions. The structure ofthe internal photosynthetic membrane systemsof whole cells has received considerable attention,and the pioneering work has been done on thefragments produced from membranes by meansof detergents. The primary phenomena of bac-terial photosynthesis take place on the uniquemembrane system of the photosynthetic bacteria,and future work will directly relate to the generalbiochemical and physical properties of thesemembranes, with emphasis on the relation of iontransfer across membranes to the electron transferand energy transfer processes which take placewithin the membrane proper. One can predictthat studies conducted over the next few yearswill yield much information about the structureof the photosynthetic apparatus at the molecularand multimolecular level. Small, photoactiveparticles have been isolated from a number ofbacteria (52-55). As described below, the isolationof a reaction center complex [containing thebacteriochlorophyll (Bchl) molecules whichutilize the absorbed light energy to initiate theelectron transfer reactions] has now been ac-complished from Rhodopseudomonas spheroideschromatophores (103), and this important eventwill most certainly be followed by incisive ex-
1Contribution no. 320 of the Charles F. KetteringResearch Laboratory, Yellow Springs, Ohio.
periments on the exact nature of the reactioncenter of the bacterial photosynthetic system.
I will not attempt to give a comprehensivereview of all aspects of bacterial photosynthesis,but rather will restrict myself to the photochem-ical electron transfer reactions which take placein the membrane system, and attempt to relatethese to the energy transfer reactions and struc-ture of the membrane. Other sources that may beconsulted as general references for the process ofbacterial photosynthesis are found in references20, 26, 27, 56, 65, 100, 119, and 122.
PRMARY PHOTOCHEMICAL REACTION
Pigment SystemsThe photosynthetic bacteria comprise three
groups which are distinguished by the nature ofthe pigment (chlorophyll) they contain and thenature of the substrates they utilize. The greensulfur bacteria, or Chlorobacteriaceae, containchlorobium chlorophyll and small amounts ofbacteriochlorophyll-a, and their metabolism iscentered primarily around sulfur compounds;hence, they are found in nature in environmentswhich contain H2S and are illuminated. Thegenera Chlorobium and Chloropseudomonas arefound in this group. The purple sulfur bacteria,or Thiorhodaceae, contain Bchl a or Bchl b, aresulfur-oxidizing bacteria, and are representedby several species of Chromatium and Thiospiril-lum. In addition to their ability to utilize sulfurcompounds, the photosynthetic sulfur bacteriaalso metabolize organic compounds quite readily.The nonsulfur purple bacteria, Athiorhodaceae,
contain Bchl a or Bchl b, and are primarilydedicated to the metabolism of organic com-pounds. Examples of the latter group are thegenera Rhodopseudomonas, Rhodospirillum, andRhodomicrobium.The chlorophylls occur in the photosynthetic
bacteria in different combinations (91). Bchl ais found in most purple bacteria as the majorpigment, and is also found in small amounts inseveral strains of the green bacteria. Structurally,Bchl a is quite similar to chlorophyll a of greenplants. Bchl b is found in only a few purple bac-teria and in a green photosynthetic rod (46, 64).The green bacteria have as the primary pigment acharacteristic chlorophyll known as Chlorobiumchlorophyll, of which several closely relatedtypes occur in a single bacterium along with asmall amount of Bchl a.
In the purple bacteria containing exclusivelyBchl a, the pigment occurs in the cell in differentforms, which are recognized by the differentabsorbance maxima shown by the intact cell.Bchl a in organic solvents shows an absorptionmaximum in the region of 770 nm, whereas in thecellular environment absorption bands are notedin the regions of 800, 850, and 880 nm. TheBchl forms absorbing at these wavelengths arereferred to as B800, B850, and B880. The differ-ence in the absorption properties of Bchl inorganic solvent and in vivo is due to the differentenvironment in the cell, in which the Bchl iscomplexed either to a lipoprotein or to a carote-noid in the membrane, or is present as an ag-gregate of Bchl. In 1952, it was shown in thelaboratory of Krasnovskii (72) that Bchl in solidfilms or in colloidal aggregates exhibited ab-sorbance properties similar to those in the intactcells. Further work in 1967 (14, 73) concerned theeffect of solvent vapors upon the Bchl absorbanceproperties, and this work showed that the bandsobserved at 800 and 860 nm for Bchl films arealtered and a new band at 900 to 920 nm ap-peared in the presence of vapors of ether andanother solvent such as water or methanol. Thus,it is possible to change the absorbance propertiesof isolated Bchl aggregates in a manner similar tothat observed in vivo, which shows the sensitivityof the absorbance maxima of Bchl to the en-vironment in which it is contained. The Krasno-vskii group relates these data to an aggregatedform of Bchl in vivo, since they can in partduplicate the absorbance phenomena with Bchlaggregates in vitro.
Chlorophyll-carotenoid interaction has beenproposed by some as the cause for the absorbanceshifts observed for Bchl in vivo (12, 60), but thereis recent evidence against this explanation. Itappears that the carotenoids influence the shifts
only in the sense that lipids in general would havethis effect, and no specific carotenoid-Bchl com-plexes are involved. Vernon and Garcia (120)have isolated a carotenoid-protein complex fromR. rubrum chromatophores through the actionof pancreatin and Triton X-100. This complexappears to be formed during the process of diges-tion, however, for the characteristic absorbancedoes not occur in the original membrane system.
There have been three protein-Bchl complexesisolated from the photosynthetic bacteria. Onewas obtained from the green bacteria Chloro-pseudomonas ethylicum and Chlorobium thio-sulfatophilum by Olson (92). This complex con-tains Bchl a (the lesser component in these bac-teria, whose main chlorophyll is Chlorobiumchlorophyll) and shows an absorbance maximumin the infrared at 809 nm. This complex has beenextensively studied by Olson, who has suggestedthat it might function in the bacteria as a reactioncenter complex, or at least be related to the reac-tion center ofthe bacterium. The small concentra-tion of the Bchl a in these bacteria, combined withthe longer wavelength absorbance of the Bchl,is consistent with this role, but to date no de-finitive photochemical role for this complex hasbeen assigned. Two other Bchl-protein complexeswere isolated by Vernon and Garcia (120) fromR. rubrum by means of enzymatic digestion(pancreatin) in the presence of the detergentTriton X-100. One of the complexes was blue andexhibited absorbance maxima at 922, 835, 580,and 372 nm. The other complex was green andhad absorbance maxima at 780, 587, and 360 nm.The green complex (but not the blue one) isactive in simple photochemical electron transferreactions. Both are soluble in water. Because ofthe prolonged treatment with the digestiveenzymes required to form these complexes, it isdoubtful that they represent the situation as itexists in the bacterial membrane. However, theydo provide soluble complexes with which theproperties of complexed Bchl can be extensivelystudied.
Reaction CentersIn 1952, Duysens (40, 42) showed that il-
lumination of the purple photosynthetic bacteriacaused a change in absorbance at 890 nm. Thisdecrease in absorbance, or bleaching, was ac-companied by another shift in the spectrumaround 800 nm. These reactions have beenstudied in great detail by Duysen's group and byClayton (25, 128). In 1960, Arnold and Clayton(2) reported that the reaction at 890 nm pro-ceeded at 1 K, showing that thermal movementof the molecules was not involved in the reaction;rather, it must take place in a tight, well-ordered
complex. The Bch] responsible for the bleaching,and through which the photochemistry of thesystem is initiated, is called reaction centerBchl, abbreviated as P890 indicating that it isthe photosynthetically active pigment whichabsorbs at 890 nm.
There is abundant evidence that P890 in R.rubrum (it is P870 in Rhodopseudomonas sphe-roides) is a Bchl in a special environment whichin some manner confers upon it the property ofbeing very reactive in electron transfer reactions,and which allows it to function as the initiatorof the electron transfer reactions peculiar tophotosynthesis. Light energy absorbed by otherBchl molecules is transferred to the reactioncenter Bchl for the initial photochemical event,which involves the transfer of an electron fromthe P890 to some adjoining molecule. The energycontained in the light quantum is thus transferredto the membrane in the form of chemical energywhich is expressed by the distribution of electronsbetween the various compounds on the mem-brane. Therefore, we are immediately concernedwith the oxidation and reduction reactions ofthese components, and it becomes the problemof the investigator to follow these reactions anddeduce the sequence of electron transfer reactionswhich follow the absorption of the quantum oflight.The early experiments of Duysens and the
later ones of Clayton showed a change in aBchl which absorbs at 800 nm when chromato-phores are illuminated. (The term "chromato-phore" refers to the small vesicular membraneunits which are obtained after rupture of thecell by either sonic oscillation or grinding. Thephotosynthetic machinery is contained on thesesmall units which are apparently formed duringdisruption of the internal membrane system ofthe cell.) Clayton (30) has studied this change inR. spheroides, and has shown that it belongs to aBchl molecule which is in a special relationshipto the P870. The 800 nm form (called P800)always occurs in conjunction with the P870, and,when the bulk of the Bchl is destroyed by oxida-tion with iridic chloride, the resultant prepa-rations show only the P800 and P870 (29). Theband at 800 nm is 2.4 times as intense as the oneat 870 nm. Illumination causes reversible bleach-ing (oxidation) of P870, which represents theinitial electron transfer reaction of bacterialphotosynthesis. Using differential extractiontechniques, Clayton (29) showed that the treatedchromatophores contained two molecules ofP800 for every P870 molecule. The spectra of suchchromatophores are shown in Fig. 1. Absorbedradiant energy is transferred more efficientlyfrom the P800 to P870 than is light absorbed by
zJ0
-j
a.0
4 -
2
0
0-8-
0 6
0*4
0.2
0-500 600 700 800 900
WAVELENGTH, m/L
FIG. 1. Absorption spectra of potassium iridicchloride-treated chromatophores ofRhodopseudomonasspheroides, with and without actinic illumination,showing the presence of both P800 and P870 and thephotooxidation of the latter in the treated chromato-phores. After Clayton (28).
the bulk of the Bchl, which indicates a closeassociation between the two special Bchl forms.When a light quantum is absorbed by Bchl in
the internal membrane system of a photosyntheticbacterium, the energy of the light quantum istransferred to the Bchl system, but is not localizedin one particular Bchl. Rather, it is contained in afunctional unit consisting of about 40 moleculesof Bchl in the case of R. rubrum (28, 83), and thisunit is serviced by one reaction center, P890.The absorbed energy activates the P890 to initiatean electron transfer reaction with surroundingmolecules, one of which is an electron donor andanother is an electron acceptor. The net resultof the photoreaction is the transfer of an electronfrom the donor to acceptor, as discussed in asubsequent section.
Quinones as Electron Acceptors
There is good evidence that quinones functionas electron acceptors in bacterial systems. Themost common quinone in the photosyntheticbacteria is ubiquinone, which occurs with sidechains of different lengths. Ubiquinone-10 isfound in Rhodopseudomonas capsulata, R. pa-lustris, R. spheroides, and Rhodomicrobiumvannielii, whereas Rhodopseudomonas gelatinosacontains the quinone with eight isoprene unitsin the side chain, ubiquinone-8 (21). Chromatiumstrain D contains ubiquinone-7 (50), Chromatium8379 contains ubiquinone-10 (21), and C. vinosumcontains ubiquinone-8 (95). The concentration ofubiquinone varies in these organisms, but it isalways a major component, reaching as high asone quinone to three Bchl molecules in the caseof photosynthetically grown Rhodospirillum
rubrumn (21). Furthermore, the quinone concen-tration is higher in the bacteria (Athiorhodaceae)when they are grown photosynthetically. Anotherquinone, rhodoquinone, is found in R. rubruin(59, 96, 111) and in Euglena (101). This quinoneis structurally quite similar to ubiquinne,with the methoxy group on the 2 position re-placed by an amino group (80). Takamiya et al.(111) have examined a large number of photo-synthetic bacteria and algae, and they havereported that ubiquinone is the major quinonein all Athiorhodaceae examined, and that R.rubrumn also contains rhodoquinone. Chronatihuinstrain D contains vitamin K9 in addition toubiquinone and the green sulfur bacteriumChloropseludomnonas etllylicumn contains vitaminK2 and a Chiorobiuni quinone-like substancePreliminary experiments performed in ourlaboratory (125) indicated that, in the case ofRhodopseudonionas palustris and R. viriclis (origi-nally described as Rhodopseudonionas speciesNHTC 133), a vitamin K-like substance is alsopresent.
Because of their ubiquitous occurrence in highamounts in the photosynthetic bacteria, andbecause of their ready interaction with photo-excited chlorophyll in solution (106). quinonesare very good candidates for interaction as anelectron acceptor with reaction center Bchl. Thisreaction can be detected from absorbance changesin the ultraviolet region which result from theabsorption of actinic light by the Bchl. Investiga-tion of whole cells does reveal absorbance changesin the appropriate regions, but the direction ofthe changes indicates that the quinone may beeither reduced or oxidized when the cell is il-luminated. Redfearn (102) studied the redoxstate of ubiquinone in R. rubruml cells and chro-matophores by extracting the cells and deter-mining the extent of reduction of the quinone inthe extract, and found that illumination causedan increase in the amount of the oxidized form.Parsons (98) measured the changes in absorbancefor R. rubrum cells and showed that, whereasillumination caused a slow oxidation of theendogenous hydroquinone, the addition ofphenylmercuric acetate changed the nature of theresponse so that a rapid reduction was observedupon illumination. Working with Clhr-omatium,Takamiya and Takamiya (110) observed thatunder aerobic conditions the absorbance at 275nm decreased, indicating a reduction of thequinone; this reduction amounted to about 7',of the total quinone. Adding Na2S203 enchancedthe change so the 25 of the quinone becamereduced. Illumination under anaerobic conditionsin the presence of malate, in which case the
quinone would be more reduced prior to il-lumination, caused an oxidation of the hydro-quinone form, which amounted to 50%- of thetotal.Whereas illumination of whole cells may cause
either an oxidation or a reduction of the quinone,experiments performed on isolated chromato-phores by following absorbance changes haveroutinely shown only a reduction of the endoge-nous quinone. Working with chromatophorefilms from Chroiniaitini, Rhodlospfirill/un rubruni,and Rhodopseudoinonas sp)heroidles, Clayton (24)showed that illumination caused absorbancechanges in the ultraviolet region correspondingto the photoreduction of ubiquinone, whichappeared to be coupled to the photooxidation ofendogenous cytochrome. Also, Baltes aLnd Vernon(5) have demonstrated a photor-eduction ofubiquinone coupled to the oxidation of the dye2,6-dichlorophenol-indophenol (DPIP) by R.rubruin chromatophores. The reactions of en-dogenous quinone in R. rubruln chl-omatophoreswhich had been treated witlh ferricyaLnide tooxidize the bulk Bchl have been determined byBeugeling (13), who showed a close relationshipbetween the photoxidation of the reaction centerP890 and the absorbance change thought toreflect the reduction of quinone. The amounits ofP890 oxidized and ubiquinone reduced uponillumination were approximately equal, and hadsimilar quantum requirements (0.8 to 1.3 quantaper molecule). Similar results w-ere obtained byKe et al. (66) on a small fragment produced fromChrolnatiaUnn chromatophores by the action ofTriton X-100. This fragment contains the re-action center P890 and endogenous ubiquinone.and illumination causes an oxidation of the P890and an apparent reduction of the quinone in acoupled reaction (Fig. 2). In this case, however,six quanta are required for the oxidation of oneP890 molecule or the reduction of one ubi-quinone. The primary nature of the reaction isindicated by the fact that it proCeeds at thetemperature of liquid nitrogen. The light-minus-dark difference spectrum shows only changescorresponding to P890 oxidation and ubiquinonereduction, and addition of ascorbate and phen-azine methosulfate (PMS) at high concentrations(which allows the reduced PMS to react withoxidized P890) in the light results in a steady-state difference spectrum corresponding toubiquinone alone. This is an important datum.since it shows that the absorbance change ob-served in the ultraviolet region with a trough at275 nm is not due to P890 oxidation and is mostlikely due to ubiquinone. Recent experimentationwith improved instrumentation on the same
FIG. 2. Light-induced absorbance chatsponding to photooxidation of reaction XP890, and reduction of ubiquinone (275small fragments producedfrom Chromatiunphores through the action of Triton X-1OGH refers to the heavy fraction obtainedgradient centrifugation in sucrose, whichparticle containing the reaction center; t,to the light fraction prepared in the samefraction is photochemically inactive. Se66for more detail.
particle from Chromatium shows thaction times of the P890 and ubiqiequally fast, and are as fast as 1 Ms(personal communication).The data cited above indicate that
reduction of endogenous quinone couloxidation of reaction center Bchl, buitwo data that are not explained byinterpretation. The first problem discu,related to the fact that both quinoneand reaction center Bchl oxidation havquantum requirement in the systems iito date. This would not be expectedquinone change is a two electron reacP890 oxidation involves only one elesecond problem relates to the fact that tspin resonance (ESR) signal observe(bacterial species investigated cannotto a quinone radical. This would in(the transition from the radical formthe fully reduced form (UQH2) is(less than 1 msec). If the second elecfrom a molecule other than the Bchl,ence in the quantum requirementsexplained. Much more work needs tothis important area.
All the data obtained for ubiquinor
n/i. are consistent with the quinone acting as anelectron acceptor for the reaction center Bchl.This reaction could be a direct one, or some
<> unknown intermediate compound could inter-H vene. Since the P890-ubiquinone coupled reaction
with Chromatium particles proceeds at 77 K,the quinone and P890 must be closely coupledin order for the electron transfer to take place in
L the frozen system. It is still possible, however,that some other intermediate compound couldintervene. To date, however, the known com-pound which functions closest to the reaction
H center Bchl as an electron acceptor is ubiquinone.Chromatophores of the photosynthetic bac-
teria (R. rubrum, Chromatium, R. spheroides) arealso capable of reacting photochemically withadded exogenous quinones, as shown by theexperiments of Zaugg et al. (134). With ubiqui-none-2 or ubiquinone-6 as the electron acceptor
nges corre- and the reduced forms of DPIP, PMS, or N,N,-center Bchl, N',N'-tetramethyl-p-phenylenediamine (TMPD)nm) in the as the electron donors, very rapid electron transfern chromato- reactions were observed upon illumination. A). The letter similar reaction is observed when reduced mam-by density malian cytochrome c is used as the donor mole-
is the small cule (133). These experiments show that thehe L refers isolated chromatophores have the ability toway. The L couple with added ubiquinones with shorter!e reference side chains (which are more water-soluble), but
the reaction is also catalyzed by isolated Bchlat the re- which is solublized by the presence of asolectinainone are (134). Further experiments of this type wereec (B. Ke, performed by Redfearn (102), who showed that
the reaction was not specific for ubiquinonethere is a derivatives, since other benzoquinones werepled to the also active in the reaction with reduced cyto-t there are chrome c. Later experiments by Zaugg et al.this simple (135) showed that the PMSH2-ubiquinonessed above photoreaction is not coupled to adenosine tri-reduction phosphate (ATP) formation, and therefore does
re the same not involve the endogenous electron transfernvestigated components of the chromatophore which aresince the involved in the cyclic electron transport system
.tion while which couples to ATP formation. Since thisctron. The reaction would not be limited in rate by the usualhe electron dark enzymatic steps encountered in the in vivod with the system of the chromatophore, it would be ex-be related pected to proceed at high rates, and would alsodicate that not be coupled to ATP formation.(UQH) tovery rapid Ferredoxintron came Another compound which is a candidate forthe differ- the role of electron acceptor for the reactioncould be center Bchl is ferredoxin. In plants, ferredoxin
be done in serves the role as the first stable photoreducedproduct, and it in turn leads to the reduction of
ne changes nicotinamide adenine dinucleotide phosphate
(NADP) (104). In the chloroplast system, how-ever, there is good evidence that another com-pound, called X, functions between ferredoxinand the reaction center chlorophyll a, P700(45, 70, 123), and its photoreduction has escapeddetection to date. In bacteria, also, ferredoxinappears to be a normal constitutent of the photo-synthetic system. It has been detected in R.rubrum (109), has been isolated and crystallizedfrom Chromatium (4), and has been purifiedfrom R. palustris (132) and Chlorobium thio-sulfatophilum (47). The protein isolated fromR. palustris has most of the properties of a fer-redoxin, but has a higher midpoint potentialthat the other ferredoxins isolated from thephotosynthetic bacteria. It appears to be inter-mediate between the ferredoxins and the high-potential iron proteins isolated from Chromatium(39) and R. gelatinosa (37).To date, it has not been possible to show a
requirement for ferredoxin isolated from aphotosynthetic bacterium for the photoreductionof nicotinamide adenine dinucleotide (NAD)by that bacterium. Experiments have beenperformed with R. rubrum (85) and Chromatium(62), but the ferredoxin from R. palustris wasnot tested in a photochemical system (132).Therefore, there is no direct evidence of a role forbacterial ferredoxin in NAD photoreduction.The ferredoxin from Chlorobium thiosulfato-philum, however, is reduced photochemicallyand supports the fixation of carbon dioxide intopyruvate and a-ketoglutarate (48) in a newlydiscovered system of carbon dioxide fixation inthe photosynthetic bacteria.
Recent studies have shown that 2-amino-4-hydroxy pteridines are associated with the photo-synthetic apparatus in a number of bacteria(51, 79, 86). Since the presence of inhibitors ofpteridine formation also causes an inhibition ofphotophosphorylation and carbon dioxide fixa-tion in these organisms (86), it is possible thatthe pteridines could serve as electron acceptorsfor the reaction center Bchl. Their low potentialmakes this hypothesis attractive, since in thereduced form they could transfer electrons toeither ferredoxin or ubiquinone, but to datethere has been no definitive evidence for such arole. Isolated bacterial chromatophores do reactwith a number of artificial electron acceptors,however, including oxygen (75, 114), quinones(133, 134), methyl viologen (coupled to a disulfideas the final acceptor) (82), and methyl red (3).
Cytochrome c as Donor for Reaction Center BchlIn 1953, a cytochrome c was observed in R.
rubrum (113, 115), and Duysens observed that
this cytochrome became oxidized in the light(41). Since that time, numerous cytochromes ofthe c type have been isolated from the photo-synthetic bacteria (11), and it is apparent thatcytochromes of this type are found in all photo-synthetic bacteria. Cytochromes of the b typeare also present, and it is commonly held thatthese cytochromes have the same relationship inthe chromatophores that they do in other electrontransfer systems; i.e., they form an electrontransfer couple which functions in the photo-synthetic cyclic electron transfer system whichleads to ATP formation (119, 124). Whereas inmitochondrial electron transfer systems the cyto-chrome c transfers electrons to cytochromeoxidase and thence to oxygen, in the photosyn-thetic bacteria cytochrome c transfers the electronto photooxidized P890. Examples of photo-oxidation of cytochrome c are too numerous tobe discussed in detail here. Some representativepapers on the subject are discussed in reference121.The cytochromes are photooxidized with
quantum requirements of 0.7 to 1.4 for Chroma-tium and 3 to 4 for R. rubrum (1). This indicatesa very efficient reaction for the oxidation of theendogenous cytochrome. Evidence that the cyto-chrome and P890 form a complex that efficientlytransfers an electron from the cytochrome to theoxidized P890 was obtained by Chance andNishimura (22), who showed that with Chroma-tium the photooxidation of cytochrome 423.5(one of four cytochromes oxidized) proceeds at77 K with an efficiency about equal to that atroom temperature. The cytochrome was notreduced during a subsequent dark period. Onthis basis, it was proposed that the primaryphotoreaction of bacterial photosynthesis wasthe transfer of an electron from the complexedcytochrome to the reaction center Bchl. A sub-sequent study by Vrendenberg (130) has shownthat, with other bacteria studied, cytochromephotooxidation decreased as the temperature waslowered, and the photooxidation stopped atdifferent temperatures. Apparently, the highlyefficient cytochrome oxidation shown by Chroma-tium is unique, and these data indicate that cyto-chrome oxidation is not necessarily the firstreaction of photosynthesis. This will be discussedin more detail below.
Since the original discovery by Olson (88) thatcytochromes in chromatophores of Chromatiumare oxidized by light, there has been intenseinterest in this organism. Based on a series ofstudies by his group, Duysens (45) has concludedthat in the case of Chromatiumn there are two cyto-chromes of the c type involved in the photo-
chemical reactions, but these serve differentfunctions. One of these, called cytochrome C232.5(the number indicates the maximum for the light-induced difference spectrum), functions in thecyclic electron transfer system characteristic of allphotosynthetic bacteria, while the other functionsin the terminal system through which electronsare fed from the substrate molecules into thephotochemical system. The two cytochromes areexperimentally distinguished by their differentreaction kinetics and by their oxidation rates inair. Similar experiments with R. rubrum havebeen performed by Sybesma and Fowler (108),who also propose the presence of two photo-systems corresponding to these two functions ofthe cytochrome c. Also, by determination ofaction spectra for the photooxidation of the twocytochromes of Chromatium, Morita (81) showsthat the various forms of Bchl in this bacteriumare effective to different degrees in causing theoxidation of these cytochromes. From thesedata, he proposes the presence of three separatephotochemical systems in this organism. Cusa-novich et al. (36) have studied particle prepa-rations from Chromatium, and have proposed theexistence of two separate electron transfer systemsin this bacterium, one driven by P890 and anotherdriven by a reaction center Bchl absorbing at905 nm. Thus, it is evident that the cytochromesof the c type can be experimentally distinguishedaccording to function in the photosyntheticapparatus, and it appears that these cytochromesare also in different environments as far as theBchl system is concerned.The oxidized form of the reaction center Bchl,
P890, reacts readily with electron donors otherthan endogenous cytochrome c. The experimentsof Zaugg et al. (133, 134) show that the reducedforms of PMS and TMPD couple efficiently withoxidized P890. Further evidence relative toTMPD is available from experiments relating tothe light-induced ESR spectrum (61), and ex-periments showing the effect of PMS on eitherthe light-induced P890 absorbance changes orthe ESR signal show that the reduced form of thiscompound donates electrons directly to oxidizedP890 (33, 66, 97).
Primary Photochemical Act
As discussed above, two consequences of theabsorption of a light quantum by the photo-synthetic systems of bacteria are the subsequentreduction of endogenous ubiquinone and theoxidation of endogenous cytochrome. Which ofthese is the primary act? Considering the quinoneas an acceptor (A) and the cytochrome as a
donor (D), the following two possibilities exist:
(1) D *P890-A ) D*P890+ *A-- D+ P890 A--
(2) D.P890*A hg D+P890-*A-- D+*P890A-
In case 1, the photoexcited P890 reacts initiallywith the acceptor to produce an oxidized P890,while in case 2 the P890 reacts first with the donormolecule to produce a reduced P890. The avail-able evidence indicates that mechanism 1 aboveis the correct one, for the following reasons.
Bleaching of the reaction center Bchl, P890,represents an oxidation of this Bchl moiety, sincesimilar absorbance changes can be brought aboutby the addition of oxidizing agents such as fer-ricyanide to the chromatophore system (25, 27).A reduction of the P890 could also cause a loss ofabsorbance of P890, but there is no direct experi-mental evidence that such a reaction takes place.One of the initial responses of the bacteria!
photosynthetic system to light is the productionofsome compound with unpaired electrons, whichresults in the observations of a light-inducedESR signal. According to Loach et al. (76, 77),this light-induced ESR signal is related to theoxidation of P890 in a one-electron oxidation, atleast in the case of R. rubrum chromatophores.An extensive study of the light-induced ESRsignal from Chromatium cells and chromato-phores conducted by Schleyer (105) led to theconclusion that the ESR signal was produced bya component of the photosynthetic electrontransfer system of this bacterium. The signal wasshown by the organized photosynthetic systemsin both the cell and the chromatophore, and wasalso shown by Bchl in solution. The compoundwhich is responsible for these light-induced re-sponses shows a midpoint potential of 0.44 v(76) which is in agreement with its function as thereaction center Bchl. In the oxidized form, itcould receive electrons from the reduced cyto-chromes whose potentials generally fall in therange of 0.29 to 0.33 v (11).The characteristics of interaction of phenazine
methosulfate with illuminated chromatophoresare consistent with its interaction with the oxi-dized form of P890. Cost et al. (33) have reportedthat this compound causes an increase in thedecay rate of the photo-produced ESR signal andthe related absorbance change. The data of Zaugget al. (134) show that the reduced form of PMSreacts with both reaction center Bchl and Bchlin solution.The data of Ke et al. (66) show that with the
small particle isolated from Chromatium chro-matophores the absorbance changes related to
the photoreduction of endogenous ubiquinonetake place in a coupled reaction with P890. Theexperiments reported allowed a comparison ofthe rise times of these reactions, from which itwas concluded that they were faster than 50 msec.Later experiments (B. Ke, personal coinmnunication) show that these reactions are both fasterthan 1 ,sec. There is no cytochrome oxidationwith this particle, and when the reduced form ofPMS is added the decay rate of the P890 absorb-ance change is drastically increased, so that athigher PMS concentrations there is no observableabsorbance change at 890 nm. At the same time,the absorbance changes due to ubiquinone con-tinue. These data indicate a direct transfer ofelectrons to the quinone as the first photoreaction.
Definitive data on the initial photochemicalreaction are furnished by the experiments ofParsons (97, 99), who used a 30-nsec flash froma ruby laser to initiate the photochemical events.With Chromatium chromatophores, the oxidationof P890 was accomplished in less than 0.5 Asec,and the presence of dithionite abolished thereaction (presumably by reducing the acceptorquinone). The oxidized P890 recovered in 2Asec, and parallel experiments showed that thecytochrome c422 became oxidized at the same rate,indicating that the P890 donated its electron toan adjoining acceptor molecule in the initialphotochemical reaction and subsequently re-ceived an electron from the adjoining cytochromein a secondary reaction. In experiments with R.rubrumn, it was found that the initial reaction was
complete in less than 0.2 ,usec. Thus, in bothbacteria, the oxidation of P890 is the primaryevent.
In considering the compounds which mightserve as the electron acceptor for photoexcitedP890, ubiquinone and ferredoxin are the logicalcandidates. The latter is reduced photochemicallyby Chlorobiumn thiosulfatophilum (48) and isutilized in carbon dioxide reduction. The factthat a number of the photosynthetic bacteriaevolve hydrogen gas upon illumination on ap-propriate substrates (119) shows that the bacteriacan operate at the low potential required forhydrogen evolution. However, to date there is noevidence that ferredoxin can be reduced in adirect reaction with the reaction center Bchl.There is evidence that ubiquinone is reduced in areaction that is primary (13, 66). This will bediscussed in more detail below.
SECONDARY BIOCHENIICAL REACTIONS
Once the initial photochemical step is ac-complished, the subsequent reactions are bio-chemical in nature and consist of a series ofelectron transfer reactions through a complex ofcompounds contained in the bacterial membranesystem. Accompanying this electron transferprocess is the formation of ATP; the process iscalled photophosphorylation. In principle, then,this system should be like the membrane systemof mitochondria and of chloroplasts, which alsocarry out electron transfer reactions and an as-sociated phosphorylation. Figure 3 presents in
Cyt c
a~~~~~~ATP
Light P800 - pBCI P890 Cyb
"*..Trans-02 hydrogenase| , +~~~~~~~~~2
t 't I~~~~~~~~X Quinone - Quinone fi-- Succinate
Ferredox inlll NxAD l
Photochemical System Aerobic System
FIG. 3. Electron transfer pathways in Rhodospirillum rubrum. The movement of electronts through the cyclicpathway, driven by the initial photochemical reaction of P890, produces the high eniergy state, X- I, which maybe used as indicated. The photoreduction of NAD which is observed with isolated chromatophores is driven bythe high-energy state, andfor reasons given in the text is portrayed as resultintg from the aerobic metabolic systemwihich is present in this bacterium.
schematic form the components of the bacterialelectron transfer system, but more specifically itpertains to R. rubrutm. The scheme shows acyclic system containing the Bchl in three forms,the bulk Bchl, the reaction center P890, and theP800 which always accompanies the P890. Thissystem serves to capture the light energy andinitiate the electron transfer process by theprocess described above. In addition to the Bchlsystem, the cyclic system is shown as containingubiquinone, cytochrome b, and cytochrome c,as well as an unknown compound X which servesas the initial electron acceptor. There is not spacein this review to consider the evidence placingthe cytochromes in this cyclic system, and thereader is referred to references 119 and 121 formore information. The positions of cytochrome cand ubiquinone are discussed above, and X isincluded in the scheme for the following tworeasons. In experiments currently underway withthe small particle obtained from Chromatiumby the action of Triton X-100 (66), under someexperimental conditions it is possible to observea light-induced oxidation of P890 with no ac-companying observable reduction of the ubiqui-none, indicating that some other compound isserving as the electron acceptor. Secondly, in thecase of Chlorobium thiosulfatophilum, ferredoxinis photoreduced by the chromatophore system,showing that it is possible to photoreduce thislow-potential compound in a system which doesnot photoreduce NAD, making it highly likelythat the ferredoxin is reduced by direct electrontransfer from the Bchl system via X. Ferredoxinis not listed as the initial acceptor, since in allexperimental procedures tried to date, in whicheither Bchl or Chl has been used, ferredoxin doesnot act as an electron acceptor for the photo-excited chlorophyll in a manner similar to qui-nones. Furthermore, all evidence points towarda similar situation in the plant chloroplast, wherethe P700 chlorophyll a photoreduces somecompound prior to ferredoxin (123).
Also shown in Figure 3 are the components ofthe aerobic electron transfer system which isfound in R. rubrum cells. This bacterium has theability to grow in the dark by virtue of an oxida-tive metabolism, although this supports a ratherslow growth and only low rates of oxygen utiliza-tion are observed. This system is depicted asbeing coupled to the photochemical cyclic systemin two ways: via reversible electron transferreactions between the quinones and by the high-energy state produced during energy transferreactions leading to ATP formation. It is notknown whether these two systems are containedon the same membrane system, or whether they
are on separate systems. The coupling of the twosystems via the high-energy state, X-I, is dis-cussed below in relation to the mechanism ofNAD photoreduction. The separate nature ofthe quinones in the two systems is illustrated bythe fact that the difference spectrum obtainedfor the reduction of endogenous quinone bylight differs from that obtained by the chemicalreduction with succinate (98). The photoreductionofNAD by this bacterium could be accomplishedeither by using succinate to feed electrons intothe system as shown or by using other donors(including artificial ones such as DPIP or TMPD)to feed electrons into the photochemical system.
NAD PhotoreductionIn those cases where a photoreduction of a
nicotinamide nucleotide has been shown, NADis the nucleotide reduced preferentially by bac-teria. Although a photoreduction of NAD hasbeen shown for Chromatium chromatophores, itis limited and has not been studied to the extentthat the reaction in R. rubrum has (62). A photo-reduction of NAD with chromatophores ofRhodopseudomonas capsulata has been reportedby Klemme and Schlegel (69), with rates up to50 Aumoles per hr per mg of Bchl reported. Thisphotoreduction resembles that observed for R.rubrum, in that suitable donors for the reactionare succinate, reduced DPIP, and hydrogen. Theeffect of added ferredoxin on this reaction wasnot reported. In the case of R. capsulata (69),R. spheroides (94), and R. rubrum (112), NADPis also photoreduced, but this reaction is coupledvia a transhydrogenase to the initial photo-reduction of NAD.The photoreduction of NAD has been studied
most extensively with R. rubrum cells and chro-matophores. A more detailed review of the earlierexperiments of this nature is given in references119 and 118, so these experiments will be givenonly a cursory treatment here. Duysens andSweep (43) and Olson et al. (89) followedNAD photoreduction in R. rubrum and Chro-matium cells by means of the increase in bluefluorescence which results from this reaction.Duysens (44) and Amesz (1) extended thesestudies with R. rubrum and R. spheroides wholecells, showing that a high rate of NAD photo-reduction was observed for short periods afterthe onset of illumination, giving a rate of 360,umoles of NAD photoreduced per hr per mg ofBchl. This rate is considerably faster than thatobserved for intact chromatophores, which atbest give rates of 50 (same units).The photoreduction of NAD by isolated chro-
electron donors which can interact with the chro-matophore electron transfer system. Frenkel(49) used reduced flavin mononucleotide (FMN)as the donor, whereas in the early experiments ofVernon (116, 117) succinate was used as thedonor. Nozaki et al. (84, 85) used ascorbate andDPIP as the donor system in their series ofexperiments. The simplest interpretation of thephotoreduction observed with chromatophoreswas that the electron donor (succinate, DPIPH2,etc.) was donating electrons for the electrontransfer system, and the electrons were thentransferred via photoexcited Bchl to the NAD.This was the interpretation placed on the reactionby the early investigators, and was used as thebasis for Nozaki et al. to distinguish betweencyclic and noncycic photophosphorylation inthe bacterial system by analogy to the plantsystem. Gest (16, 57) proposed that the so-callednoncyclic photophosphorylation in R. rubrulnwas actually of the cyclic type, and was proceedingsimultaneously with the net electron transferfrom the donor to the acceptor, and that, further-more, the cyclic electron transfer system func-tioned in the reaction to produce ATP whichthen drove the electron transfer reaction in an
energy-requiring step. Thus, with succinate as
the donor, the succinate-to-NAD electron transferis an energy-requiring reaction, and the energy
for the step is provided by the ATP produced bycyclic photophosphorylation.
Recent experiments on isolated R. rubrurnchromatophores have shown that at least some,
and most likely all, of the observable NADphotoreduction proceeds via the reverse electronflow mechanism. The most convincing datumsupporting this concept is the demonstration ofa succinate-supported reduction of NAD bychromatophores in the dark when ATP is sup-
plied. Low and Alm (78) reported in a 1964abstract that this reaction had been noted, but itwas described in detail by Keister and Yike (68).Although the rate of the dark ATP-driven re-
action is low by comparison with the photo-reduction (we have never observed it to exceedone-third of the light-induced rate), the effectsof inhibitors of electron transport and uncouplersare consistent with the concept that both go bysimilar mechanisms. Uncouplers of phosphoryla-tion inhibit both reactions, whereas inhibitorsof electron transport peculiar to the cyclic electrontransport system inhibit only the light-drivenreaction. Oligomycin stimulates the light-drivenreaction while it inhibits the ATP-driven reaction.The addition of a phosphate acceptor system(adenosine diphosphate plus inorganic phos-phate) inhibits the light-driven reaction, and thisinhibition is overcome by the addition of oli-gomycin.
Keister and Yike propose that the NAD re-duction, both the light-driven and the ATP-dependent type, is accomplished by the aerobicelectron transport system found in R. r-ubrunicells. It is for this reason that Fig. 3, which wasadapted from that presented by Keister andYike (68), shows the NAD reduction system asseparate from the cyclic electron flow system.This would be consistent with the fact thatappreciable rates of NAD photoreduction areobserved only with bacteria which have an aerobicmetabolism, such as R. rubruln and R. cap)suIlata(although R. spleroides does not show NADphotoreduction). The photoanaerobe Cliromia-tium, which shows a photoreduction of NAD inexperiments conducted with intact cells, showsonly a very low rate of NAD photoreductionwith chromatophores, and this could be relatedto the very slight aerobic metabolism carriedout by this organism (58).
Since the rate of NAD photoreduction byintact R. rubrum cells is far greater than is ob-served with isolated chromatophores, and sincethat observed by the chromatophores is mostlikely carried out by the aerobic metabolismcomponent of the bacteria, we must still con-sider the possibility of a direct photoreduction ofNAD by the photochemical system of the bacte-ria, which could be routinely inactivated throughthe process of rupturing the cells and preparingthe chromatophores. The ability of Chlorobiurnthiosulfcatophiluni chromatophores to photoreduceferredoxin, and the inability of other photo-synthetic bacteria to do so, although ferredoxinis present in the cells, indicates that this abilityis probably lost or masked during the prepa-ration of chromatophores of these bacteria.
Trebst et al. (112) have studied the effect of aninhibitor of ferredoxin reduction for plant chloro-plasts, disalicylidenepropanediamine disulfonicacid, upon the photoreduction reactions of R.rutbrurn. Whereas this inhibitor is effective withplant chloroplasts, it has no effect upon NADphotoreduction by R. Erubr uti chromatophores.Also, the addition of ferredoxin to the NADphotoreduction system has no effect. These dataare in accord with the conclusion that the NADphotoreduction of R. rubrum involves the aerobicsystem, and it is driven by the high-energy state,X-l, formed by cyclic electron flow as shownin Fig. 3.Another important reaction involving the
nicotinamide nucleotides in bacterial photo-synthetic systems has recently been reported byKeister and Yike (67), who demonstrated thatR. rubrumiii chromatophores have an energy-requiring transhydrogenase (the enzyme whichtransfers electrons between NADH and NADP),and that this enzyme can be activated either by
light (which produces the high-energy state viaelectron transfer reactions) or by the addition ofATP or of pyrophosphate. The equivalence ofATP and pyrophosphate in R. rubrum chromato-phore reactions will be discussed below. A similarreaction has been reported for R. spheroideschromatophores by Orlando et al. (94), but inthis case pyrophosphate could not be used as anenergy source to drive the reaction.
Photophosphorylation and Energy TransferSince the formation of ATP accompanying the
photosynthetic electron transfer reactions isalmost universally observed, it is reasonable toassume that it occurs in all photosynthetic sys-tems, and, in cases where it is difficult to demon-strate, some operational obstacle for its exhibitionis present. There is considerable literature on thesubject of bacterial photophosphorylation, andthe interested reader is referred to previousreviews and articles for background material(16, 56, 57, 85, 119). The outline of the process isshown in Fig. 3, which indicates that there aretwo sites of ATP formation in the cyclic electrontransfer system. The presentation of two sitesfor ATP formation is based on some early ex-periments from the laboratory of H. Baltscheff-sky. In collaboration with Arwidsson (7), heobserved that the antibiotic valinomycin inhibitedATP formation in R. rubrum chromatophores tothe extent of only 50%, which they interpretedto mean that one of two phosphorylation siteswas available to valinomycin.A second reason for thinking in terms of two
phosphorylation sites is the fact that the inhib-itors antimycin A and HOQNO affect the cyclicelectron transport system of R. rubruin chro-matophores and completely stop the cyclicphosphorylation process. The addition of PMSor DPIP to the inhibited system allows phos-phorylation to proceed again (see reference 119for pertinent references). Since PMS is thought toreact with oxidized P890 (33, 66, 97, 135) andthe inhibitors are thought to act at the cyto-chrome b level, this means there must be a phos-phorylation site on the electron transfer spanwhich is not bypassed by the PMS, or at thequinone level. This is substantiated by the ex-periments of Baltscheffsky et al. (6), who reportedthat the quantum requirement for ATP forma-tion in R. rubrum chromatophores was approxi-mately 6 quanta per ATP formed; in the presenceof antimycin A and PMS, the requirement wentup to about 10 quanta per ATP formed. Thesedata indicate that one site is inhibited and non-functional in the presence of antimycin A, andthat the other site is carrying out the ATP forma-tion under these conditions. The site functioning
under these conditions is presumably located atthe quinone level in Fig. 3. Further evidence forseparate sites of phosphorylation in R. rubrumchromatophores is provided by the experimentsof Horio and Yamashita (63), who showed thatirradiation with ultraviolet light or treatmentwith Triton X-100 distinguishes between thephosphorylation catalyzed by added ascorbateand that stimulated by added PMS.Another expression of the energy-conservation
mechanism of R. rubrum chromatophores is theformation of pyrophosphate, P-P, coupled tophotosynthetic electron transport (8, 9). In thisreaction, the activated phosphate intermediateis transferred to a phosphate molecule instead ofadenosine diphosphate, so that pyrophosphateresults. This reaction represents another way inwhich the activated state produced by electrontransport may be energetically utilized; also, theuse of pyrophosphate provides another way ofproducing the high-energy state in bacterialchromatophores. M. Baltscheffsky et al. haveused both ATP and pyrophosphate to producethe high-energy state in R. rubrum chromato-phores, and have shown that this is then utilizedto cause a reduction of cytochrome b (9, 10).Addition of these compounds causes an increasein absorbance at 423 nm, which is interpreted tomean a reduction of cytochrome b. These dataindicate that cytochrome b is involved in ATPformation in this bacterium, and would locateone of the two phosphorylation sites at thecytochrome b level, or at the cytochrome b-cytochrome c couple. This site would be the oneinactivated in the system inhibited by antimycinA in the presence of PMS. Using the same tech-nique, absorbance changes corresponding to thecarotenoid compounds can be observed, whichwould indicate that the transformation responsi-ble for the carotenoid shift is also intimatelyrelated to the phosphorylation mechanism.
Following the observation that light causes anuptake of protons into chloroplasts, bacterialchromatophores were examined to see whether asimilar reaction occurred. Subsequent to theearly observation of Chance et al. (23), othershave studied this reaction (34, 126, 127). Themagnitude of the reaction observed with R.rubrum chromatophores is less than that observedfor spinach chloroplasts. The quantum yield ofproton movement into the chromatophore is atbest 0.1 proton moved per quantum (23), whichis considerably less than the value of 5 protonsmoved per quantum absorbed by spinach chloro-plasts (38). On the basis of these data, Chanceet al. concluded that proton movement per sewas not involved in a direct way in the transferof energy from the electron transfer system to
ATP formation, but was rather a secondaryreflection of some phase of the ATP formation.There remains considerable work to be done
before the proton change induced by light inbacterial chromatophores can be related in amechanistic way to the electron transfer reactionsand ATP formation. Whether the proton gradientso produced is the energetic force which drivesATP formation is neither proved nor disproved.The proton movement is obviously closely relatedto the ATP-forming system, since the protongradient is strongly affected by uncouplingagents. von Stedingk and Baltscheffsky (126)reported that, whereas gramicidin inhibits theproton change in R. rubrum chromatophores,valinomycin in the presence of potassium ionstimulates the proton movement. A significantstudy made recently in the C. F. KetteringLaboratory by Shavit et al. (107) revealed thatthe antibiotic nigericin in the presence of potas-sium was a potent uncoupler of phosphorylationin R. rubrum chromatophores, and under theproper conditions it inhibited the proton pumpwhile leaving ATP formation not significantlyaffected.
STRUCTURE
There has been considerable interest withinthe past few years in the structure of the photo-synthetic apparatus in bacteria, with emphasisupon the use of detergents to fragment the mem-brane into smaller units which can be studiedfor their composition and photoactivity. Thetreatment of bacterial systems with detergentsbegan with the observations by Komen thatsodium dodecyl sulfate produced changes in thelocation and intensities of the Bchl absorptionbands of R. rubrum and Chromatium chromato-phores (71). Subsequently, Brill (17) used thedetergent Triton X-100 to convert R. spheroideschromatophores into two fractions, one enrichedin the longwave Bchl form (B880) and the otherenriched in the two shorter wavelength formsB850 and B800. (These terms denoting thelocation of the three main Bchl absorption bandsare used for all bacteria considered, although insome cases the actual absorption maxima maydiffer from these wavelengths.) Brill later studiedthe effects of deoxycholate upon Chromatiumchromatophores (18, 19) and showed that ableaching of the B850 occurred. In this case,energy transfer between the Bchl forms wasinhibited, which is another indication of a struc-tural alteration caused by the detergent. Claytonperformed experiments on Chromatium, andshowed that deoxycholate produced two frag-ments, and one was enriched in the B880 compo-nent.
We have begun a study of the fragmentation ofthe purple photosynthetic bacteria with TritonX-100, and have studied the fragments so pro-duced in some detail. A general treatment ofthese investigations has appeared (125), andmore specific papers have been published whichcontain the details for the experiments withChromatium (52), R. rubrum (53), R. palustris(54), and Rhodopseudomonas sp. NHTC 133(R. viridis; 55). (For a general treatment of theinternal structure of the photosynthetic bac-teria, see reference 32.) Chromatophores fromthe purple bacteria examined are fragmentedinto two fragments which are separable by densitygradient centrifugation. One of the fragments isa small particle, and the other one is more mem-branous in nature, indicating that the detergentremoves a portion of the photosynthetic systemin the form of the small particles, leaving theresiduum intact.A comparison of the properties of the frag-
ments produced by Triton X-100 is shown inTable 1. The fragments are designated as "heavy"or "light" depending upon their relative positionsafter sucrose density gradient centrifugation. Thesmall particle released by the detergent may beeither the light or the heavy fraction, and in some(but not all) cases there is a separation of theBchl forms between the two fragments. Electronmicrographs of the various fractions obtainedfrom these four bacteria are contained in reference125. The bacteria examined to date fall into oneof two groups. The first one, including Chroma-tium and R. palustris, have the photochemicallyactive particle in the heavy fraction, and thereis a clear separation of the Bchl forms betweenthe two fragments. Only the small particle carriesout the primary photochemical reactions, asshown by the oxidation of the reaction centerBchl and the reduction of endogenous ubiqui-none. The other group includes R. rubrum andR. viridis, which yield two fractions that do notdiffer appreciably in Bchl content. The lightfraction contains the photoactive particle, but thephotooxidation of reaction center Bchl also takesplace in the other fragment, which is more mem-branous in nature.The data obtained in our laboratory indicate
that the detergent Triton X-100 solubilizes aportion of the bacterial membrane system,liberating small particles into the medium. Inthe cases we have examined, this particle con-tains the reaction center Bchl and the long wave-length Bchl (B880). This shows a clear distinctionin the environment of the various Bchl forms inthese bacteria. The small particles liberated varyin size and in their tendency to aggregate. Inthe case of R. palustris, the particles tend to
TABLE 1. Properties of the subchromatophore particles prepared from four species of photosyntheticbacteria through the action of the detergent Triton X1OOa
Chromatophorefragment
ChromatiumHeavy
Light
Rhodopseudo-monas pa-lustris
Heavy
Light
Rhodospirillumrubrum
HeavyLight
Rhodopseudo-monasNHTC
Heavy
Light
Appearance
Particulate, 0.7pm
Membranous
Particulate, instrands 0.65 to0.80 pm wide
Membranous
MembranousParticulate, 0.5pm
Aggregated par-ticles (1.3 pmspacing)
Particles, someaggregated,1.35 pm
Bchl contentb
A 880/A 8 A 880/As86
(0.58)3.4
0.12
(1.14)
2.4
0.63
(4.7)
3.53.6
(6.7)
8.0
8.0
(0.60)3.1
0.24
(0.9)
1.3
0.56
ReactioncenterBchlc
Absent
Absent
+
+
Endoge-nous
UQ photo-reductiond
Absent
Absent
Absent
FastESRsignal
Absent
+
Quinonecontent"
UQ
UQ
UQ (6.7), vita-min K-like
UQ (5.4), vita-min K-like
UQ (2.8)UQ (3.4)
UQ (1.6), vita-min K-like
UQ (1.4), vita-min K-like
a The designation of heavy and light fractions refers to the relative position following a sucrose den-sity gradient centrifugation. UQ represents ubiquinone.bThe absorbancies (800, 850, and 880 nm) refer to the general locations of the three forms of Bchl.
The exact locations in each case were as follows: Chromatium, 890, 850, and 800 nm; R. palustris, 873,857, and 802 nm (805 in chromatophores); R. rubrum, 877 and 800 nm; and Rhodopseudomonas NHTC,1,005 (1,015 in chromatophores) and 830 nm. The values in parentheses are for chromatophores.
c The reaction center Bchl was measured at the following wavelengths; Chromatium, 890 nm; R.palustris, 870 nm; R. rubrum, 890 nm; and Rhodopseudomonas NHTC, 940 nm.
d Determined by absorbance change at 275 nm.e The numbers in parentheses are the ratios of Bchl to UQ on a molar basis. Similar data are not
available for Chromatium, but ubiquinone is present in approximately equal amounts on the two frac-tions. The vitamin K-like quinone migrates in the region of vitamin K during thin-layer chromatog-raphy, but has not been positively identified.
form predominantly linear aggregates 6.5 to8.0 nm thick. Some linear aggregates are alsofound with R. viridis, but more often the ag-gregates are of a two-dimensional planar nature.Where tests have been made, the cytochromesare found in this fraction, although it has notbeen possible to demonstrate a photooxidationof the cytochromes. Perhaps the cyclic electrontransfer system has been disrupted so that onlythe initial electron transfer from the reactioncenter Bchl to the adjoining ubiquinone moleculeis seen.
The plant chloroplast system is similarly af-fected by the detergent Triton X-100 (123, 125),yielding two fractions which are representativeof the two photosystems operative in plantphotosynthesis. Whether Triton X-100 or digi-tonin is used for the fragmentation process, thesmall particulate fraction so produced containsphotosystem 1, shows the photooxidation ofP700 (the reaction center Chl a in plants), andcarries out a photoreduction of NADP. This isthe plant counterpart of the bacterial smallparticle. In the plant system 1 particle, there is a
photochemical transfer of an electron from theP700 reaction center Chl a to some acceptor, X,and thence to ferredoxin. The analogy betweenthe plant and bacterial systems carries over tothe photooxidation of P890 and a photoreductionof ubiquinone. This points out a similarity be-tween the plant and bacterial systems in termsof their component parts and general reactions,both photochemical and biochemical. In bothcases, the complete photosynthetic apparatusincludes distinct units which are imbeddedwithin and form part of the membrane system.The membrane continuum contains the shorterwavelength chlorophyll forms, which in the caseof bacteria consist of the accessory Bchl formswhich are designed to capture the light quantaand transfer the energy so obtained to the reactioncenter where the reduction of NAD (leading toCO2 fixation) takes place. In the case of the plantchloroplasts, also, the membrane continuumcontains the shorter wavelength-form of Chl a ofphotosystem 2, which leads to the evolution ofoxygen. This photosystem is accessory, however,since its function is to provide electrons to photo-system 1 so that NADP photoreduction (andcarbon dioxide fixation) can proceed. Sincesubstrate molecules furnish the electrons neces-sary for carbon dioxide fixation in bacteria, thereis no need for the elaborate biochemical ma-chinery required to oxidize water; therefore,there is no need for the separate photoact thatis seen in plants, and the shorter wavelengthBchl serves only in a light-harvesting capacity.The successful isolation of the reaction centerfrom R. spheroides has recently been reported byReed and Clayton (103), who used Triton X-100to fragment the chromatophores into threefractions which were separable by density gradientcentrifugation on a discontinuous sucrose gradient.One of these fractions contained the reactioncenter Bchls, P800, and P870, but no B870.Another fraction was devoid of the reactioncenter Bchls but contained the light-harvestingBchl, B870. These data show that the reactioncenter exists as a separate entity in the chro-matophore membrane. Extensive studies of thecharacteristics of light absorbance changes andthe related fluorescence changes (31, 128, 129)show that the reaction centers exist separately inthe membrane and are fed absorbed light energyby the Bchl system which forms the bulk of themembrane system. The reaction centers are notindependent in terms of accepting energy, butrather form an integrated system with the light-harvesting Bchl continuum so that light absorbedby the bulk Bchl is available to a number ofreaction centers. Clayton concluded (31), "En-
ergy transfer extends over a number of P870molecules, as if the tissue contains an extendedmatrix of light-harvesting pigment studded hereand there with photochemical reaction centers."Our data are certainly in agreement with thisconcept.
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