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Primary Photochemistry in the Facultatively Aerobic Green

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Primary Photochemistry in the Facultatively Aerobic Green Proc. Natl Acad. Sci. USA Vol. 79, pp. 6532-6536, November 1982 Biophysics Primary photochemistry in the facultatively aerobic green photosynthetic bacterium Chloroflexus aurantiacus (photosynthesis/bacteriochlorophyll/reaction center/redox potentiometry/evolution) BARRY D. BRUCE*, R. CLINTON FULLER*t, AND ROBERT E. BLANKENSHIPtt *Department of Biochemistry, University of Massachusetts; and MDepartment ofChemistry, Amherst College, Amherst, Massachusetts 01002 Communicated by William Arnold, August 18, 1982 ABSTRACT Photochemical activity was examined in mem- chemistry of Chloroflexus, and in this paper we present evi- brane fragments and a purified membrane preparation from dence that the primary photoact is nearly identical to that found Chloroflexu8. Flash-induced absorption difference spectroscopy in the purple bacteria and unlike that found in other green strongly suggests a primary donor (P8m) that is more similar to the bacteria. P870 bacteriochlorophyll a dimer found in the purple photosyn- thetic bacteria than it is to PNO found in the anaerobic green bac- MATERIALS AND METHODS teria. Redox measurements on PW5 and an early acceptor also in- dicate a photochemical system characteristic of the purple bac- Preparative Methods. Organism and growth conditions were teria. The membrane preparation contains a tightly bound type the same as described by Sprague et aL (14). The isolation of c cytochrome, c5u, that is closely coupled to the reaction center whole membranes and purified cytoplasmic membranes used as indicated by its ability to rereduce photooxidized P8. Chioro- the procedures described by Feick et aL (13). flexus thus appears to be distinct photochemically from other fam- Single-Flash-Induced Absorption Change Measurements. ilies ofphotosynthetic bacteria and may occupy an important role Measurements were made on a homemade single-beam laser in photosynthetic evolution. spectrophotometer. Actinic illumination (583 nm; 1-,sec flashes) was provided by a Candela SLL-625 flashlamp-pumped dye Many ofthe electron transfer components found in the fourfam- laser with rhodamine 590 as the dye. ilies of anoxygenic photosynthetic bacteria are also character- Absorption and Difference Spectra. Absorption spectra istic ofoxygenic photosynthetic organisms. The two families of were recorded on Cary 14R or Cary 219 spectrophotometers. purple photosynthetic bacteria, Rhodospirillaceae and Chro- Absolute cytochrome difference spectrawere recordedwith the matiacae, have an electron acceptor system strikingly reminis- Cary 219 using dithionite and ferricyanide as reductant and cent ofthe acceptor found in photosystem II ofoxygen-evolving oxidant, respectively. Light-induced absorption changes in the organisms (1). In contrast, the electron acceptor system of the near infrared (>900 nm) were measured on a Cary 14R spec- anaerobic green bacteria, the Chlorobiaceae, is similar to the trophotometer in the IR-2 mode. acceptors found in photosystem I (2-6). Redox Potentiometry. Redox potentiometry was performed Little has been reported on the primary processes of pho- in a system similar to that described by Dutton (15). A double- tosynthesis in the recently discovered family ofthe facultatively bridged Ag/AgCl combination redox electrode (no. 7025-02-90, aerobic green bacteria, Chloroflexaceae. This family is unique Ingold, Andover, MA) was used; it was calibrated before each in that it shares many features of both the green and purple use by measurement of a standard redox buffer solution (In- bacteria. It resembles the Chlorobiaceae in that it contains two gold). Dithionite and ferricyanide were used as titrants. Me- types ofbacteriochlorophyll (Bchl), Bchl c and Bchl a. The Bchl diators and titrants were prepared freshly in anaerobic solutions c in Chloroflexus and all other green bacteria is contained in of either double-distilled H20 or absolute ethanol. They were antenna-like structures called chlorosomes. The reaction center kept on ice and in the dark until used. Mediators used were Bchl a in all green bacteria including Chloroflexus is located phenazine ethosulfate (PES), phenazine methosulfate (PMS), in an undifferentiated cytoplasmic membrane rather than in the N,N,N',N'-tetramethylphenylenediamine (TMPD), 2,3,5,6- highly differentiated intracytoplasmic membrane characteristic tetramethyl-p-phenylenediamine (DAD), duraquinone, and ofall purple bacteria (7-9). Metabolically, Chloroflexus resem- vitamin K. All potentials are relative to the normal hydrogen bles the Rhodospirillaceae in having the capability to exist as electrode. a light-independent aerobic heterotroph (10). EPR Spectroscopy. EPR spectra were recorded at room tem- Pierson and Castenholz (11) and Schmidt (12) have shown perature on aVarian E-9 spectrometer. Reversible light-minus- that most of the Bchl a in Chloroflexus aurantiacus has an in dark spectra were produced by subtraction of dark-after-light vivo absorption spectrum similar to thatfound in purple bacteria spectra from the light spectra. and unlike the Bchl a absorption spectrum ofthe green anaer- obic bacteria. A continuous light-induced difference spectrum RESULTS suggested the presence ofa Bchl a-type reaction center pigment The whole-membrane fraction (Fig. 1A) was derived from cells analogous to that occurring in the purple bacteria (11). grown under low light conditions (A740/Aw = 20) as defined Feick et aL (13) recently developed a procedure for isolating by Feick et aL (13). Fig. 1B is an absorption spectrum of cyto- from Chloroflexus cytoplasmic membranes that are completely plasmic membranes (CM). The low absorbance at 740 nm in- devoid of Bchl c yet still contain photochemical activity. Using these preparative techniques, we have examined the photo- Abbreviations: Bchl, bacteriochlorophyll; DAD, 2,3,5,6-tetramethyl- p-phenylenediamine; PES, phenazine ethosulfate; PMS, phenazine The publication costs ofthis article were defrayed in part by page charge methosulfate; TMPD, N,N,N',N'-tetramethylphenylenediamine; CM, payment. This article must therefore be hereby marked "advertise- cytoplasmic membranes. ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. t To whom reprint requests should be addressed. 6532 Downloaded by guest on October 3, 2021 Biophysics: Bruce et al Proc. Nati. Acad. Sci. USA 79 (1982) 6533 0 0.8 0.6 -20 w < A' 400 600 800 400 600 800 Wavelength, nm FIG. 1. Absorption spectra. (A) 'Whole membrane fraction" from cells grown under low light conditions to be used for cytoplasmic mem- brane isolation. (B) Isolated cytoplasmic membranes. FIG. 3. Reversible light-induced EPR spectra of both whole mem- branes (200 KP) and isolated CM. Both traces are light-minus-dark dicates that this fraction is largely free of Bchl c. CM prepara- after light. Modulation amplitude at 100 kHz, 4 G; receiver gain, 3.2 tions with AW/A740 ' 5 were used for this work. x 104. Fig. 2 shows the flash-induced difference spectrum of pu- rified CM at ambient redox potential. We performed a similar AHpp, of 9.8 ± 0.2 G. This g factor is consistent with a Bchl a experiment with whole membranes between 750 and 1,000 nm cation radical, and the narrow linewidth suggests that this rad- (data not shown). -Both whole membranes and purified CM ical probably consists of a Bchl a dimer (17). This EPR signal showed bleaching at 865 and 823 nm and an absorbance increase almost certainly arises from the photooxidized primary donor at 805 nm. In purified CM, a smaller photobleaching at 600 nm pigment, analogous to that found in all other photosynthetic and absorbance increases at 430 and 760 nm were apparent. organisms. At room temperature this photooxidation was almost These absorbance changes were not detectable in this region completely reversible. in whole membrane preparations because of the large absor- The results of a redox titration of PM are shown in Fig. 4. bance ofthe antenna system. At this redox potential there were This titration indicates a one-electron reaction with a midpoint no absorbance changes which could be attributed to a cyto- potential at pH 8.1 of +360 mV. As the potential was decreased chrome. The time course offlash-induced absorbance changes below +300 mV, the amount of Pwz photobleaching began to at 865 and 805 nm are shown in Fig. 2 Inset. Both absorbance decrease. This decrease in AAM at lower potentials did not changes had similar biphasic recovery kinetics. These changes involve any interactions with the mediators as shown by its in- were almost certainly due to the photooxidation ofthe reaction dependence of mediator concentration (data not shown). The center Bchl and its subsequent rereduction (16). Continuous low potential decrease in AAwz fits well to a one-electron re- light-induced changes in the infrared region showed two peaks, action with' a midpoint potential at pH 8. 1 of +255 mV. Both at 1,160 and 1,250 nm. Both peaks were completely reversible titration curves were reversible and showed little hysteresis. (data not shown). Chemically induced reduced-minus-oxidized cytochrome Light-induced EPR spectra in the g = 2.0 region are shown difference spectra are shown in Fig. 5. Both whole membranes in Fig. 3. Both the whole membrane fraction and purified CM and isolated CM from anaerobic light-grown cells showed the showed a symmetric, reversible light-minus-dark signal cen- characteristic shape of type c cytochrome (c5) with a, /3, and tered at g = 2.003 ± 0.001. Both had a peak-to-peak linewidth, Soret peaks at 554, 525, and 422 nm, respectively. The differ- ence spectrum from whole membranes of aerobic dark-grown o x < -5 -10 -15 nf% - "40t0 500 600 700 800 900 1000 500 Wavelength, nm Eh, mV FIG. 2. Laser flash-induced difference spectrum of purified CM at ambient redox potential. Each point represents the mean of 6-10 FIG. 4. Redox titration of Pw5 in isolated CM. The mediators were traces. The calculation was made by using data from 10 msec after the PES, PMS, DAD, and TMPD, each at 20 ,uM. *e Oxidative titration; flash. (Inset) Representative absorbance changes at 865 and 805 nm. A, reductive titration (both were done on the same sample).
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