Analysis ofoptical spectra fromsingle crystals of Rhodopseudomonas viridis reaction centers (dichroism spectra/crystallized reaction center/excitons/protein-pigment interaction/photosynthesis)

Absorption spectra andlight-induced ab- sorbance changes ofcrystals fromRhodopseudomonas viridis reaction centers arerecorded. Atheoretical analysis oftheab- sorption andcircular dichroism spectra ispresented, yielding aconsistent picture ofspectroscopic andstructural informa- tion. Theprimary process inbacterial photosynthesis involves a light-driven electron transfer across amembrane. Itiscata- lyzed byaprotein-pigment complex, thephotosynthetic re- action center (RC). Thechemical nature andpigment com- position areknownforanumberofRCs(1-4). TheRCfrom thepurple photosynthetic bacterium Rhodopseudomonas viridis wascrystallized (5); theRCsinthose crystals arepho- tochemically active (6). Recently, anx-ray structure analysis at3A resolution provided thedetailed arrangement ofthe pigments intheRC(7)(four bacteriochlorophyll b(BC), two bacteriopheophytin b(BP), andfourhemegroups). The hemegroups areboundtoac-type cytochrome. TheBCs andBPsareassociated withtheprotein subunits LandM, respectively, andareindexed inthis paperbyLandM ac- cordingly. Thesepigments arearranged intwobranches, which exhibit anapproximate 2-fold rotation symmetry (7). Attheaxis ofrotation, twoBCsinteract closely (Mg-Mg distance, -7A)withtheir pyrrole rings Istacked ontopof eachother. Theyformthespecial pair andaredenoted BCLP andBCMP.Nexttothespecial pair aretheso-called accesso- rymonomers BCLAandBCMA.TheMg-Mgdistance be- tweentheBCinthesamebranch is=11A.Inclose proximi- tytotheBCASarethetwoBPsattheendofthebranches. Thedistance ofthering centers ofBPandBCAfromthe samebranch is-11A.Theapproximate 2-fold rotation sym- metry isbroken bythepresence ofonly onequinone, proba- blymenaquinone, attheendoftheLbranch; apparently, the RC'ssecond quinone waslost during purification orcrystal- lization. Inthis paper, weinvestigate absorption spectra andab- sorbance changes after illumination withactinic light byus- ingsingle crystals ofdifferent orientations. Thisyields acon- siderable increase ofinformation ascompared tospectra fromRCinsolution. Circular dichroism (CD)spectra ofre- action centers insolution arealso considered (8,9).The spectra areanalyzed byusing thestructural information of theRCs(7). (Seealso figure 1inref. 10.) Materials andMethods Theisolation ofthereaction centers andthecrystallization procedure hasbeendescribed (5,6).During theinvestiga- tion, thecrystals werekeptinclosed cells containing buffer solution (6). Thincrystal platelets (-0.2 mm x0.1mm x20 sAm) withthesmallest extension inthex orydirection and (0.1 mm x 0.1mm x 20Am)withthesmallest extension in thezdirection wereoccasionally obtained bycrystallization methods. Theywereusedinthetransmission experiments, allowing absorption measurements along different crystallo- graphic directions. Thecrystals belong tothetetragonal space groupP43212. Fortetragonal crystals, itissufficient to consider twogeometries fortheabsorption measurements where theelectric field vector Eofthelight isparallel (Ell) or perpendicular (El) tothetetragonal zaxis, respectively. Ab- sorption spectra ofother geometries canbederived fromthe results ofthese twospectra. Thetransmission measurements areperformed byusing twospectrometers (resolution, 3nm),polarizing optics, a microscope, photomultipliers, andphase-sensitive detec- tion. Carewastaken tokeepthemeasuring atthelowest possible level tominimize absorption changes duetothe photochemical activity ofthereaction centers. Forthat rea- son,thefirst spectrometer ofasmall spectral bandwidth (3 nm)wasusedbefore thesample. Thesecond spectrometer just infront ofthephotomultiplier wasusedtoreject stray light andthelight oftheactinic radiation. Theactinic light source wasaXenonarclampinconjunction withaspec- trometer (960 nm < X< 980nm).Theintensity level ofthe exciting light waskeptsufficiently lowtoensure alinear de- pendence oftheabsorbance change onthelight intensity.