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Biochimica et Biophysica Acta 1507 (2001) 278^290 www.bba-direct.com

Review The antenna reaction center complex of : composition, energy conversion and electron transfer1

Sieglinde Neerken *, Jan Amesz

Department of Biophysics, Huygens Laboratory, Leiden University, P.O. Box 9504, 2300 RA Leiden, The Netherlands

Received 11 December 2000; received in revised form 26 April 2001; accepted 18 June 2001

Abstract

A survey is given of various aspects of the photosynthetic processes in heliobacteria. The review mainly refers to results obtained since 1995, which had not been covered earlier. It first discusses the antenna organization and pigmentation. The pigments of heliobacteria include some unusual : (BChl) g, the main pigment, 81 hydroxy a, which acts as primary electron acceptor, and 4,4P-diaponeurosporene, a carotenoid with 30 carbon atoms. Energy conversion within the antenna is very fast: at room temperature thermal equilibrium among the approx. 35 BChls g of the antenna is largely completed within a few ps. This is then followed by primary charge separation, involving a dimer of BChl g (P798) as donor, but recent evidence indicates that excitation of the acceptor pigment 81 hydroxy chlorophyll a gives rise to an alternative primary reaction not involving excited P798. The final section of the review concerns secondary electron transfer, an area that is relatively poorly known in heliobacteria. ß 2001 Elsevier Science B.V. All rights reserved.

Keywords: Heliobacterium; Electron transfer; Energy transfer; Bacteriochlorophyll g

1. Introduction green sulfur , were already described in the 19th century. The discovery of the heliobacteria The heliobacteria were discovered in 1984 by Gest presents us with a splendid example of a scienti¢c and Favinger [1]. Their isolation, together with that accomplishment made as a result not of planned re- of the green ¢lamentous bacteria in 1974 [2], doubled search, but of a keen observation. This was described the number of known groups of anoxygenic photo- by Gest some years ago [3]: an accidental error in the synthetic bacteria. The other two, the purple and the preparation of an enrichment medium used in a mi- crobiology class at Indiana University produced a culture with an unusual brownish-green color. The culture was not discarded, as many of us would do, Abbreviations: A0, primary electron acceptor; ARC, antenna reaction center; BChl, bacteriochlorophyll; Chl, chlorophyll; but examined and found to contain a new species of HPLC, high performance liquid chromatography; MK, menaqui- photosynthetic bacteria. The newly isolated species none; P798, primary electron donor; RC, reaction center was called Heliobacterium (Hbt.) chlorum and found * Corresponding author. Fax: +31-71-527-5819. to belong to an entirely new group of photosynthetic E-mail address: [email protected] (S. Neerken). 1 Dedicated with fond memories and thanks to my mentor and bacteria with unusual pigmentation, and with a new friend, Jan Amesz, who died on 29 January 2001 shortly after the and hitherto unknown pigment, bacteriochlorophyll completion of this review. g [4].

0005-2728 / 01 / $ ^ see front matter ß 2001 Elsevier Science B.V. All rights reserved. PII: S0005-2728(01)00207-9

BBABIO 45081 15-10-01 S. Neerken, J. Amesz / Biochimica et Biophysica Acta 1507 (2001) 278^290 279

Fig. 1. Taxonomic relationship of some species of heliobacteria. The lengths of the connecting lines are proportional to the number of substitutions in 16S rRNA gene sequence analyses. Adapted from [28]. Fig. 2. Elution pattern obtained by normal phase HPLC of an extract from Hbt. chlorum [46]. Note the di¡erent scales for the two parts of the ¢gure. Peak 1 is the carotenoid recently identi- The heliobacteria are the only photosynthetic or- ¢ed as 4,4P-diaponeurosporene [35], peak 5 BChl g. Peak 4 is ganisms that can be classi¢ed under the Gram-pos- the 132 epimer, BChl gP, which in dimeric form may constitute itive bacteria [5,6], in the so-called low GC group. the primary electron donor, P798 [34]. The polar pigment of They are taxonomically closer to the peak 6 is 81 hydroxy Chl a, the primary electron acceptor. than to the other groups of anoxygenic photosyn- thetic bacteria [5,7,8]. Their evolutionary position same activity for primary photochemistry as have has been discussed repeatedly [5,7^18]. Perhaps a isolated membranes or intact cells [32,33]. These hundred strains and species of heliobacteria have complexes, called antenna reaction center (ARC) now been isolated [17,19^28], belonging to four gen- complexes [33], contain a total of about 35 BChl g era, which appear to be closely related [20,21,27,29] molecules [33,34], two molecules of a pigment that (see Fig. 1). Most have been found in samples, was identi¢ed as 81 hydroxy chlorophyll (Chl) a and usually from rice ¢elds [1,21,22,24^26], but other carotenoid. On the basis of its absorption spectrum strains have been isolated from hot springs and mobility in HPLC (Fig. 2) the carotenoid was [23,26,28,30] or from the banks of soda lakes ¢rst thought to be neurosporene [15,31], but more [20,27]. Most seem to produce [19,23,25]. recently it was shown to be 4,4P-diaponeurosporene, This review will focus on results obtained during the last 5 or 6 years; earlier work has been reviewed [15,24,31] and will be discussed mainly as a basis for more recent experiments.

2. Antenna structure and pigments

The pigment system of heliobacteria is simple: it consists of a single pigment^protein complex that is embedded in the cytoplasmic membrane and con- tains both the reaction center (RC) and BChls that have an antenna function. Invaginations of this membrane or extramembranous organelles to accom- modate additional antenna pigments have not been observed, and it has been shown that one can obtain Fig. 3. Absorption spectra of membrane fragments from Hba. and purify detergent solubilized protein complexes mobilis at 6 K (solid line) and at room temperature (dotted that have the same pigment composition and the line). The spectra are not to scale (adapted from [41]).

BBABIO 45081 15-10-01 280 S. Neerken, J. Amesz / Biochimica et Biophysica Acta 1507 (2001) 278^290 with 30 instead of 40 carbon atoms [35]. This pig- ment has never been observed in other groups of photosynthetic organisms, but it has been found in some non-photosynthetic ones [36]. Takaichi et al. [35] suggested that heliobacteria may be unable to produce C20 precursors (see below). The pigment composition of heliobacteria is re- £ected in their absorption spectrum, as illustrated for Heliobacillus (Hba.) mobilis in Fig. 3. The band at 791 nm at room temperature is due to Qy absorp- tion by BChl g; the Qx band is located at 577 nm, whereas the band at 670 nm is due to 81 hydroxy Chl a, which we shall henceforth call Chl a 670. At room temperature, the Qy band of BChl g shows no struc- ture, but at liquid nitrogen temperature or lower, Fig. 4. Absorption spectrum of BChl g in diethyl ether (adapted three bands can be discerned, at 779, 794 and 809 from [115]). nm. The precise location of these bands may vary somewhat. They were ¢rst observed by van Dorssen et al. [37] in Hbt. chlorum and were supposed to complex) without peripheral antenna. The size of this re£ect the presence of three spectral pools of BChl `core antenna' apparently cannot be varied in re- g: BChl g 778, BChl g 793 and BChl g 808. Although sponse to external conditions. In this respect the he- the ARC complex is probably an exciton-coupled liobacteria would resemble those purple bacteria that system (see next section), the bands may in ¢rst ap- have only an LH1 antenna like Rhodospirillum ru- proximation be assigned to speci¢c BChl g mole- brum and Blastochloris (formerly Rhodopseudomonas cules. For the sake of convenience we shall therefore [42]) viridis. These bacteria too have a constant or use the above mentioned designations throughout almost constant number of about 30 BChls per re- this paper. The three pools are not re£ected in reso- action center [43]. It is somewhat surprising that the nance Raman spectra, which show a remarkably ho- heliobacteria have such a small and invariable anten- mogeneous population of pentacoordinated BChl g na, since most are supposed to live in mud or soil [38]. The primary electron donor, P798, is a dimer of [31], where little light would be available. However, BChl g, presumably of its 132 epimer (BChl gP) [34]. one should keep in mind that the actual growth con- The amount of BChl gP was found to be one per ditions of the heliobacteria are not well known. They approx. 17 BChls g, giving a BChl g to reaction can grow in the dark by anaerobic of center ratio of approx. 35 [34]. P798 shows bleaching lactate [44], which would be a commonly available upon photooxidation at 798 nm at room temperature substrate in rice . and at 793 nm at cryogenic temperature [15,31]. The BChl g is a `new' pigment, not earlier found in di¡erential extinction coe¤cient for P798 oxidation nature. Its electronic structure, and hence its absorp- has been determined in two di¡erent experiments and tion spectrum (Fig. 4), resembles those of BChls a appears to be higher than that of any primary elec- and b, but the esterifying alcohol of BChl g is farne- tron donor determined to date. Values of 180 mM31 sol (C15) instead of phytol (C20) of BChls a and b cm31 [39] and 225^240 mM31 cm31 [40] have been [6,34,35]. In contrast to BChl b, BChl g has a vinyl reported; the determination of the last value was not group at C3 on ring I as in Chl a [4,6]. The pigment based on the number of BChls g per P798. is less stable than e.g. BChl a, and by a simple re- All species examined so far have essentially iden- arrangement at ring II can be converted to Chl a tical absorption spectra [20,25,27,29,30,33,37,41], in- [6,45]. Fig. 4 shows the spectrum in diethyl ether. dicating the same pigmentation and a very similar The speci¢c extinction coe¤cients for BChl g at the 31 31 structure of the pigment system. This means that Qy maximum are 96 þ 5 mM cm (at 767 nm), they all possess a core pigment complex (the ARC 85 þ 4 mM31 cm31 (at 776 nm) and 76 þ 4 mM31

BBABIO 45081 15-10-01 S. Neerken, J. Amesz / Biochimica et Biophysica Acta 1507 (2001) 278^290 281 cm31 (at 762 nm) in diethyl ether, benzene and ace- Nuijs et al. [52] by means of absorbance di¡erence tone, respectively [46]. These numbers may be com- spectroscopy with membrane fragments from Hbt. pared to the extinction coe¤cients of BChl a: chlorum. They observed a rapid formation of the 31 31 ‡ 3 97.0 þ 0.5, 90.8 þ 0.3 and 69.3 þ 0.3 mM cm , re- charge separated state P798 A0 . However, the time spectively [47]. resolution of about 35 ps did not allow them to The main protein of the ARC complex from Hba. follow transfer of the excitation energy within the mobilis, PshA [48], has been sequenced [49] and pigment system and to the reaction center. Experi- found to exhibit a weak but clear homology to ments by van Noort et al. [53], with a higher time PsaA and PsaB of Photosystem I (remarkably, there resolution, showed that a thermal equilibrium be- is also a stretch that shows some homology to the tween the BChl g molecules is established within at Photosystem II antenna complex CP 47 [12]). There most a few picoseconds. The life time of excited an- are no indications for a second, homologous protein tenna BChl was found to be about 25 ps [32,53] and in the ARC. This means that the reaction center is its decay was attributed to the primary step of charge ‡ 3 homodimeric, as in [50]. Evi- separation, the formation of P798 A0 . Absorbance dence that the RC resembles that of Photosystem I di¡erence measurements with Hba. mobilis by Lin et also comes from photochemical studies, to be dis- al. [54], who obtained a time constant of 27 ps for cussed below. The number of pigments in the ARC charge separation, con¢rmed essentially the above complex with an antenna function is much smaller conclusions. Liebl et al. [55] studied the equilibration than in Photosystem I, but about twice as large as between the various BChls g in more detail. From a for the other Photosystem I-like reaction center com- comparison with the anisotropy decays, they con- plex, that of green sulfur bacteria [47,51]. cluded that equilibration between spectroscopically similar pigments occurs on the same time scale as spectral equilibration between di¡erent BChl g pools 3. Energy conversion in the pigment system (this situation contrasts with that in Photosystem I [56]). Initial anisotropy values larger than 0.4 sug- 3.1. Room temperature gested that the BChls g of the ARC complex form an excitonically coupled system. In contrast to the Energy conversion in heliobacteria in the picosec- older reports, it was found that the formation of ond time region was studied for the ¢rst time by the charge separated state is a biexponential process

Fig. 5. Schemes by Liebl et al. [55,63] of energy transfer between the di¡erent BChl g pools and trapping by P798 (A) at room tem- perature and (B) at 20 K. B778, B793 and B808 denote BChl g 778, 793 and 808, respectively. The circular arrows refer to energy transfer between individual pigments of the same pool. Reprinted with permission from [55,63], copyright 1996 and 1997 American Chemical Society.

BBABIO 45081 15-10-01 282 S. Neerken, J. Amesz / Biochimica et Biophysica Acta 1507 (2001) 278^290 with time constants of 5 and 30 ps. For further de- formed by Liebl et al. [63]. The results are summa- tails we refer to Fig. 5A. rized in Fig. 5B. Upon excitation of the short wave- One might ask if the above time constants repre- length pool BChl g 778 very fast downhill energy sent the `true' rate of charge separation. Basically, transfer was observed to BChl g 793 with a time two models could be used to explain the observa- constant of V50 fs. Excitation transfer from BChl tions. According to the so-called transfer-to-trap-lim- g 778 to BChl g 808 occurred in 400 fs and appeared ited model [57,58], the time constant of about 25 ps to be faster than from BChl g 793 to BChl g 808 would represent energy transfer from the thermally (V1.4 ps). Apparently, at least some of the BChls equilibrated antenna to P798, after which a rapid g 793 are located further away from BChl g 808 (or charge separation starting from excited P798, oriented less favorably for energy transfer) than P798*, would occur. The other extreme is the so- BChl g 778. Energy transfer among pigments of called trap-limited model [59], where P798 is part one pool occurred with time constants of about 300 of the thermally equilibrated system, with rapid ex- fs, but much slower processes were also observed in change of excitation energy between P798* and the the BChl g 808 pool (see below). excited antenna BChls g. The measured rate of Once the excitation energy has arrived in BChl g charge separation is then a function of the true 808 it is transferred to long wavelength forms of this charge separation rate and the overall equilibrium pigment as indicated by a gradual shift of the bleach- constant between the excited antenna and P798*. ing of the band. Lin and coworkers reported a time The latter depends on the e¡ective size of the anten- constant of 15 ps for this process [62,64], but a faster na. Using the trap-limited model, Lin et al. [54] cal- transfer rate of 7 ps was observed by Liebl et al. [63] culated an intrinsic time constant for charge separa- and Neerken et al. [65]. The latter measured in addi- tion of 9 1.2 ps. From the more recent experiments tion a much slower component of 120 ps [65]. These by Liebl et al. [55], however, one has to conclude data indicate a strong spectral inhomogeneity of the that spectral equilibration is not entirely completed BChl g 808 pool. As ¢rst observed by Lin et al. [62], before charge separation occurs, which implies that excited BChl g 808 decays in 30 ps at 140 K and the trap-limited model may only be an approxima- about 70 ps at 20 K. This decay was ascribed to tion. Until recently, a discrimination between the two energy transfer to P798, followed by charge separa- models was not possible. We shall return to this tion. Time constants similar to those mentioned point later. above were obtained from hole burning measure- ments in the spectral range of 780^815 nm [66]. 3.2. Cryogenic temperature Perhaps the most important conclusion of Liebl et al. [63] was that energy transfer to the primary elec- At low temperature the di¡erent BChl g pools can tron donor P798 can also occur directly, in a few be well distinguished since their optical transitions picoseconds, from BChl g 778* and BChl g 793* are clearly separated as mentioned earlier: the ab- and does not necessarily proceed via BChl g 808* sorption spectrum of BChl g in the Qy region shows (Fig. 5B). This implies that spectral equilibration is three bands due to BChl g 778, BChl g 793 and BChl not complete before charge separation takes place. g 808, respectively (Fig. 3). Energy transfer in mem- These ¢ndings are in contrast with those by Chiou branes of heliobacteria at low temperature has been et al. [64] who concluded that all excitations accumu- repeatedly studied by time-resolved absorbance dif- late on BChl g 808 before charge separation can ference spectroscopy. Already the ¢rst experiments occur. The observations by Liebl et al. [63] were showed that the excitation energy is rapidly accumu- recently con¢rmed by experiments of Neerken et al. lated on the long wavelength forms of BChl g 808 [65]. As illustrated in Fig. 6, at 10 K excitation at 770 [60]. Time constants of between 1 and 2 ps at 15^20 or 793 nm resulted on the one hand in rapid energy K were reported for the transfer of energy from BChl transfer to BChl g 808 and on the other hand in fast g 778 and BChl g 793 to BChl g 808 [61,62]. Experi- charge separation from excited BChl g 793, with a ments with a time resolution of 30^60 fs and selective time constant for the combined reaction of V1 ps. excitation of the BChl g pools were recently per- Energy transfer to P798 thus competes with energy

BBABIO 45081 15-10-01 S. Neerken, J. Amesz / Biochimica et Biophysica Acta 1507 (2001) 278^290 283

temperature from excited BChl g 808 is quite surpris- ing and the mechanism of this process is not under- stood. The long wavelength antenna absorbs near 808 nm while the transition of the primary electron donor P798 is located around 793 nm [68]. Thus, with a value for kT of 16 cm31 at 20 K, which corresponds to a di¡erence of about 1 nm for optical transitions near 800 nm, P798 would thermally not be accessible from excited BChl g 808 at cryogenic temperature. Therefore, one would expect charge separation to be a very slow and ine¤cient process [63]. Nevertheless, the yield of charge separation, even at 6 K, is still signi¢cant [69] and appears to be not much less than at room temperature [65]. One explanation for the charge separation starting from the excited long wavelength antenna, BChl g 808*, has been mentioned by Chiou et al. [64]; they sug- gested a charge separation with BChl g 808 as pri- mary reactant: P798 BChl g 808* A0CP798 BChl g ‡ 3 ‡ 3 808 A0 CP798 BChl g 808 A0 . Fig. 6. Time-resolved di¡erence spectra of membrane fragments of Hba. mobilis at 10 K upon excitation at 793 nm at various 3.3. An alternative pathway for charge separation delays after the onset of the pulse. Spectra measured at 25 ps and longer delays were shifted vertically. The spectrum at V1 ns gives the signal averaged over the time range of 650^1200 Recently, evidence was obtained for a direct path- ps. The ¢gure illustrates: (i) fast energy transfer from excited way of charge separation from excited Chl a 670 in BChl g 793 to BChl g 808; the decay of the bleaching around reaction centers of green sulfur bacteria, not involv- 793 nm occurs with about the same time constant (V1 ps) as ing excited antenna BChl [70]. However, it was not the formation of the bleaching band around 808 nm; (ii) fast clear if the trap-limited or transfer-to-trap-limited charge separation from excited BChl g 793: part of the excita- tions on BChl g 793 appear to result in rapid formation of model applied, and therefore no unambiguous con- P798‡, which also causes a bleaching band at 793 nm; (iii) clusion was possible concerning the nature of the equilibration within the BChl g 808 pool toward the red-most pathway. As described above, for the ARC complex pigments, as evidenced by a red shift of the BChl g 808 bleach- of heliobacteria an approx. 1 ps component of ing band; (iv) slow charge separation from excited BChl g 808 charge separation is observed at cryogenic, but not (time constant V500 ps); a signi¢cant increase of the bleaching around 793 nm occurs between 115 ps and V1 ns, together at room temperature. Since it is hard to imagine that with a decay of the BChl g 808 band. Adapted from [65]. the 1 ps reaction would only occur at low temper- ature, this strongly indicates that at room temper- ature the trap-limited model, or a model approaching transfer to BChl g 808. Charge separation can also it, applies. At low temperature, so far no discrimina- occur via excited BChl g 808, but this step was found tion was possible between the two trapping models. to be very slow, with a time constant of about 500 ps Neerken et al. [65] performed a comparative study [65], which is in good agreement with calculations of with excitation of BChl g and Chl a 670 which Kleinherenbrink et al. [67], based on the yield of showed that also in heliobacteria a direct pathway photochemistry. A 60 ps decay component of excited for charge separation from excited Chl a 670 exists, BChl g 808 was also observed [65], but this compo- without involving excited `antenna' BChl g. Excita- nent was not re£ected in the formation of P798‡, tion, at room as well as at low temperature, of Chl a indicating that it represents some loss process for 670 resulted in the generation of a signi¢cantly larger the excitation energy. amount of P798‡ relative to the signal of excited The occurrence of a charge separation at cryogenic BChl g than did direct excitation of BChl g (Fig.

BBABIO 45081 15-10-01 284 S. Neerken, J. Amesz / Biochimica et Biophysica Acta 1507 (2001) 278^290

was speculated that the reaction center would be similar to that of Photosystem I and green sulfur bacteria [74], and this speculation was soon sup- ported by various lines of evidence. Much of this evidence has been reviewed earlier [15,31]: (i) the homology, albeit limited, of PshA with the corre- sponding proteins PscA and PsaA and B; (ii) the smallest complex contains pigments with antenna function in addition to the reaction center; (iii) the presence of functional iron^sulfur centers in the re- action centers; (iv) the nature of the primary electron acceptor, which is a Chl a. As mentioned already, the ¢rst to observe the pri- Fig. 7. Time-resolved di¡erence spectra at 275 K upon excita- mary charge separated state were Nuijs et al. [52] tion at 770 or 668 nm. Solid lines, spectra of excited BChl g at 1.4 ps delay (normalized); dotted lines, spectra of P798‡ aver- who found, by pump probe spectroscopy, together aged over the time range of 650^1200 ps. The amount of with the bleaching due to P798‡, a bleaching near P798‡ produced relative to the number of excited states gener- 670 nm, ascribed to reduction of the primary electron ated in BChl g is about 50% larger upon excitation at 668 nm acceptor A0. Fourier transform infrared (FTIR) dif- than at 770 nm. This suggests the existence of a direct pathway ference spectroscopy of P798 indicates that the pos- for charge separation upon excitation at 668 nm without in- ‡ volving the excited BChl g antenna (adapted from Neerken et itive charge on P798 is delocalized over both BChls al. [65]). g of the dimer [75], as was earlier concluded from the width of the EPR band of the radical [73]. A0 was shown to be 81 hydroxy Chl a [46] (Chl a 670). This 7). Since, as argued above, a trap-limited model ap- assignment is supported by spectral changes in the plies for charge separation at room temperature, this Soret region [76], which resembled that of the pri- e¡ect can only be explained by a mechanism such as mary electron acceptor of Photosystem I, also be- ‡ 3 3 P798 Chl a 670*CP798 Chl a 670 . This means lieved to be Chl a [77]. Reoxidation of A0 by the that Chl a 670, which is normally the primary elec- next electron acceptor occurs in 600 ps at room tem- tron acceptor (A0), would now in the excited state perature [52,54,60,76,78] and in 300 ps at 15 K [60]. function as initiator of the photochemical reaction. A Absorbance changes near 670 nm observed at longer similar scheme for charge separation, but starting times [52,65,68,79] may be attributed to electrochro- from the accessory BChl (BA) has been proposed mic shifts of the Chl a 670 absorption band. Under for purple bacteria [71]. Other schemes, in which strongly reducing conditions, forward electron trans- 3 the excitations from Chl a 670 would ¢rst be trans- fer beyond A0 is blocked [69,76] and a 17 ns back ferred to P798 before charge separation occurs, can- reaction with P798‡ occurs, resulting in the forma- not explain the larger amount of P798‡ generated tion of the triplet of P798 by radical pair recombi- upon excitation of Chl a 670, since in a trap-limited nation [69,80]. The same time constant was derived model P798* is in equilibrium with the excited anten- from measurements of delayed £uorescence [81]. na BChl g and the excitations would rapidly di¡use Kinetic evidence indicates that there are four con- back into the antenna. secutive electron acceptors in the membrane, includ- ing A0 [39]. However, the nature of the second elec- tron acceptor, called A1 in Photosystem I, is not yet 4. Electron transfer clear. Membranes of heliobacteria contain signi¢cant amounts of menaquinone (MK) [32,82,83], mainly 4.1. The acceptor side MK 9, and the same is true for the isolated ARC complex, which contains about one molecule of MK As soon as the absorption di¡erence spectrum of 9, and small amounts of MK 8 and MK 10 [32]. P798 photooxidation had become known [72,73] it EPR, ENDOR and TRIPLE resonance measure-

BBABIO 45081 15-10-01 S. Neerken, J. Amesz / Biochimica et Biophysica Acta 1507 (2001) 278^290 285

ments [84,85] show that the quinone is reduced upon the putative terminal electron acceptors, FA and FB. illumination. However, there is no clear evidence that Optical evidence for their existence is lacking; apart the quinone is an obligatory intermediate in the elec- from indirect, kinetic arguments, the evidence is tron acceptor chain, as it appears to be in Photo- based on EPR measurements published in 1985^ system I of plants and cyanobacteria [77,86]. Flash- 1990, and mentioned already in earlier reviews induced absorbance di¡erence spectra of membranes [15,31]. The most comprehensive study can be found of Hba. mobilis resembled that of Fx reduction in in the now 10 year old publication by Nitschke et al. Photosystem I but gave no evidence for the reduction [91]. of a quinone [76,78]. Photovoltaic measurements likewise suggested no other acceptors than A0 and 4.2. The donor side and cyclic electron transfer Fx [78]. In this context it is of interest to note that 3 ‡ electron transfer from A0 to the next acceptor is The immediate electron donor to P798 is a cyto- about 20 times slower (600 ps) in heliobacteria than chrome c, cyt c553. In whole cells of Hba. mobilis 5^6 in Photosystem I [87,88]. Extraction of menaqui- hemes c and 1^3 hemes b per RC were found, and nones had no observable e¡ect on the electron trans- almost all of those were connected to the pool of ‡ fer kinetics in membranes of Hbt. chlorum [83], and it electron donors to P798 [98,99]. The cyt c553 pool should be noted that similar observations have been is inhomogeneous, and consists of at least four cyto- made recently with detergent-solubilized prepara- chromes with di¡erent midpoint potentials, ranging tions of green sulfur bacteria. These preparations from 390 to +190 mV [98,99], and di¡erent EPR lack menaquinone but do not appear to have im- and optical spectra. Half-times of cytochrome paired electron transfer [47,89,90]. oxidation of 110 and 600^700 Ws were observed Information on iron^sulfur centers as electron ac- [98,100], but the reaction slows down to the ms ceptors comes from EPR [73,84,91,92] and optical time range in broken cells and isolated membranes spectroscopy [40,93]. The primary structure of [39,72,73,94,98]. C-type cytochromes of 50, 29 and PshA indicates the presence of an iron^sulfur center 18 kDa have been puri¢ed from Hba. mobilis [99]; like Fx in the reaction center [49], and optical di¡er- the 29 kDa cytochrome may be part of the cyto- ence spectroscopy indicated that Fx is the ¢rst elec- chrome bc complex (see below). Both the 50 kDa tron acceptor after A0, and is reduced in 600 ps and the 18 kDa cytochromes c553 are discussed as 3 ‡ ‡ [40,76]. The back reaction between Fx and P798 candidates for being the immediate donor to P798 occurs in 20 ms [40,94]. Spin-polarized EPR spectra [99,101]. The gene coding for the 18 kDa cytochrome in the X and K band that could be assigned to the c553 of Hba. mobilis was for the ¢rst time sequenced ‡ 3 charge pair P798 Fx have been studied by van der by Lee et al. [102]. Membrane-bound c-type cyto- Est et al. [95]. It was shown that the observed polar- chromes of 42, 32 and 18 kDa were found in Helio- ization patterns could be reproduced assuming direct bacterium gestii [101]; the last one was puri¢ed and electron transfer from A0 to Fx with a time constant has a midpoint potential of +215 mV [101]. The gene of 600 ps [96]. At low temperature the re-reduction sequence of the 18 kDa cytochrome c553 of Hbt. ges- of P798‡ is considerably faster (2.3 ms) [68]; a sim- tii [101] showed a high degree of similarity to that of ilar e¡ect has been observed in the other Photosys- Hba. mobilis [102]. tem I-like reaction centers of green sulfur bacteria Menaquinones have an approx. 150 mV lower re- [97]. In contrast to heliobacteria and green sulfur dox potential than ubiquinones or plastoquinones bacteria, the re-reduction of P‡ in Photosystem I [103^105] and this may make menaquinone especially slows down upon cooling [77]. In this connection it suitable to function in the heliobacterial cytochrome may be noted that large di¡erences were also found bc complex, since this has to operate at a signi¢- in the rates of forward electron transfer [39,87,88], cantly lower redox potential than the corresponding indicating that there may be rather fundamental dif- complex in purple bacteria. A cytochrome bc com- ferences between the electron acceptor chain of Pho- plex has been isolated from Hba. mobilis [106,107], tosystem I and heliobacteria. and it was shown that, in whole cells, the cytochrome To our knowledge, there is no recent evidence on c553 hemes mediate electron £ow from the cyto-

BBABIO 45081 15-10-01 286 S. Neerken, J. Amesz / Biochimica et Biophysica Acta 1507 (2001) 278^290

heme iron proteins, that have been found in the green sulfur bacterium limicola [113,114], and in various other species of bacteria including archaebacteria (see [111]). Their role, if any, in photosynthetic electron transfer is not known. The ferredoxins were observed to support photoreduction of NADP‡.

5. Concluding remarks

As has been noted before [15,31], the heliobacteria as well as the green sulfur bacteria o¡er several ad- vantages over cyanobacteria, algae and higher plants Fig. 8. Scheme of electron transfer in Hba. mobilis according to for the study of Photosystem I-type reaction centers, Kramer et al. [108]; Fd, ferredoxin. Reprinted with permission from [108], copyright 1997 American Chemical Society. in particular when applying optical spectroscopy. Moreover, for many studies with heliobacteria deter- gent solubilization and fractionation are unneces- chrome bc complex toward the reaction center sary; almost all of the experiments mentioned in [98,108]. The cytochrome bc complex of heliobacteria this review have been done with membrane frag- resembles more the b6 f-type complex of oxygenic ments obtained by simple disruption of the cells. photosynthesis than the bc-type complex of purple We should be aware, however, that there are still bacteria. An extensive discussion on the evolution important gaps in our knowledge. The most impor- and relationship of cytochrome bc complexes can tant of these concerns the peptides associated with be found in the recent review by Schu«tz et al. [109]. the reaction center and the electron acceptor chain. Nevertheless, the cytochrome bc complex of helio- Only one peptide is known, and has been sequenced: bacteria has some unique properties that distinguish PshA, the only peptide found in the isolated ARC it from other ones [108], like the midpoint potential complex [32,33]. Genes of a peptide analogous to of the Rieske 2Fe2S center, which is +150 mV for PscB of green sulfur bacteria have not been found Hba. mobilis [106] and +120 mV for Hbt. chlorum [8], nor has the peptide been isolated or even ob- [110] and a similarly low midpoint potential of the served by gel electrophoresis. With regard to the ac- b cytochromes [108]. The complex showed the usual ceptor chain, little progress has been made during sensitivity to speci¢c inhibitors like stigmatellin and recent years: the existence and identity of A1 are still dibromothymoquinone (DBMIB) [108]. A scheme by in doubt, our knowledge of the iron^sulfur centers is Kramer et al. [108] of electron transfer in Hba. mo- scanty, and, except for the ¢rst steps, from P798 to bilis is shown in Fig. 8. A0 and hence to the next acceptor, the rates of elec- In a recent comprehensive study [8] for the ¢rst tron transfer are not known. Nevertheless, in general time a major photosynthetic gene cluster from a he- we now know as much about the reaction center and liobacterium was characterized, and the relationship secondary electron transfer in heliobacteria as in the with genes from other, photosynthetic and non-pho- green sulfur bacteria, but in heliobacteria this knowl- tosynthetic organisms, established. The study cov- edge has been obtained with considerably less e¡ort. ered the one known reaction center gene pshA, var- ious cytochromes and components of the cytochrome bc complex and enzymes for BChl and carotenoid References biosynthesis.

Finally it should be mentioned that a rubredoxin [1] H. Gest, J.L. Favinger, Heliobacterium chlorum, an anoxy- [111] and two ferredoxins [112] have been isolated genic brownish-green bacterium containing a `new' form of from Hba. mobilis. Rubredoxins are small non- bacteriochlorophyll, Arch. Microbiol. 136 (1983) 11^16.

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