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P-E3-18 Early charge separation in modified bacteriorhodopsins
E3 Electron transfer and energy transduction P-E3-18
P-E3-17 kJBPICQSBCOND&PBCTROSCQPY OF THE PIItYTOAFrE[VE YI%L@%V BALTUSKA A, VAN STOKKUY I KROON A,* MONSYOUWBR R. HELLINGWERF KJ, VAN GRONDELLE R.’ ‘Faculty of Physic8 and Astronomy, VrijeUniversiteit, Amsterdam Amsterdam.
(NL). Dept. of Microbiology, Amsterdam (NL)
R&ch CentreHung.Acad.Sci.,‘JATEUniv., Szeged(Hungary),‘WcizmatlnInst.,Rehovot(Israel)
‘Biol.
Univ. of
The primary eventsof the bactexiorhodopsin (bR) photocycle,studied in previousultrafast absorption kinetic experiments,can be summan‘sedas follows:
Purpose: Elucidation of the primary stepsof the photocycleof the photoactive yellow protein from Ectothiorhodospira halophila, containing a new chromophore: thiol ester-linkedp-coumaric acid. Methods: Using a laser systemof <200 fs pulse duration, basedon a Ti:S pumped optical parametric generatorand a white light continuum generator, transient absorption was measuredat 20 wavelengths(from 430-550 nm) with excitation at 400 and 460 nm. The data were globally analysed. Results: A satisfactoryfit required three components. Next to a non-decayingcomponent, the lifetimes were 0.61 and 2.5 ps (400 nm excitation) and 0.78 and 3.8 ps (460 nm excitation). All DecayAssociatedSpectra(DAS) showed bleachingand absorption. In addition, the first and secondDAS exhibited stimulated emission. Conclusions: The first intermediate state of the PYP photocycleis formed in -0.6 ps and hasa lifetime of -3 ps. Despite the different chromophores, obtained lifetimes strikingly resemblethoseof early intermediatesof bacteriorhodopsin,appearing due to chromophore photoisomerization.
The time resolution (-3 ps) presentlyavailable for protein electricresponsesignal (IJERS)of bR is not enough to assignits early components to this scheme. Here we report ultrafast PERSdata on (i) bR mutants with considerablyprolonged lifetime of the excited stateand (ii) bR samplesof artificial retinal pigment with blocked trans-cisisomerization.
P-E3-19 REDUCTION OR11Y. Dept.of PublicHealth.Graduateschoolof Med. KyotoUniversity (Japan) Purpose: Full descriptionof the spectraland redox behaviorof hemeaA (Fe,,) and heme aB(Fee) that deservesmechanisticanalyses. Methods: Cytochromec oxidasepurified from bovine he&t musclewas reducedwith sodium dithionite underanaerobicconditionsin a stoppedflow rapid-scanappqatus. The spectralchanges recordedfor 30 min were subjectedto data analyses by global fitting to derivekinetic parameters. Results: Basedon a reactionmodel consistingof 11 specieswith specificspectralfeaturesthe observed spectralchangeswere reproducedsati&factorily, thusrigorousanalysesof the reactionmechanism being enabled. Conclusions:The redox potential of FeAis higher than that of Fesby 80 mV. This relationshipand the intrinsicabsorptionspectraof Fe,,end Fesin both oxidizedand reducedstatesare unchanged throughout the reductionprocess.The redox potentialsof both Fe,,and Fea are indep&dent of the redoxstatesof CUB . Thus aqy assumptionlike a ‘ncodasdiqiimoUeI’is neededto a&am rather comp?ica&ispectralchanges&ainrd here.
‘Div. of Chemistryand Cl&i&al Enginee&g, CALTEC, Pasad= (USA), ‘Dept. of Bi Univ. of California at San Diego, La Jolla,9 (7J A) Purpose: To investigatethe relation between the geometricand electronic structuresof the binuclearcenter of CuAin qtochrome oxidase and to study the effect of H&II) on the metal center. Methods: S-, X- and Q-band EPR was used combined with sim+tions and CNDO/S calculationson a 109~atomsystem. Results and ca~~cluslaas: The two Cu ions were nearly identical with gx = 1.996, gv= 2.011, g, = 2.187,,IA+15 G, wd = 29 G and /.4d= 28.5 G. Thelm~w@hwas&depcndentinamanuer ttqimbpy in the A-tensor. ?iI%zt$ &FL IS calculations,basedon the X-ray structureof the Paracoccus dmihijrcons ve, yielded a s’ y ocmqied au&ding orbatalE.a~hCu is A*To ndedtooneSand a’-bQuded to&e other. wast%lmdto extrude one of the Co torrs, T saving the other in the reduced state. 162
P-E3-17 kJBPICQSBCOND&PBCTROSCQPY OF THE PIItYTOAFrE[VE YI%L@%V BALTUSKA A, VAN STOKKUY I KROON A,* MONSYOUWBR R. HELLINGWERF KJ, VAN GRONDELLE R.’ ‘Faculty of Physic8 and Astronomy, VrijeUniversiteit, Amsterdam Amsterdam.
(NL). Dept. of Microbiology, Amsterdam (NL)
R&ch CentreHung.Acad.Sci.,‘JATEUniv., Szeged(Hungary),‘WcizmatlnInst.,Rehovot(Israel)
‘Biol.
Univ. of
The primary eventsof the bactexiorhodopsin (bR) photocycle,studied in previousultrafast absorption kinetic experiments,can be summan‘sedas follows:
Purpose: Elucidation of the primary stepsof the photocycleof the photoactive yellow protein from Ectothiorhodospira halophila, containing a new chromophore: thiol ester-linkedp-coumaric acid. Methods: Using a laser systemof <200 fs pulse duration, basedon a Ti:S pumped optical parametric generatorand a white light continuum generator, transient absorption was measuredat 20 wavelengths(from 430-550 nm) with excitation at 400 and 460 nm. The data were globally analysed. Results: A satisfactoryfit required three components. Next to a non-decayingcomponent, the lifetimes were 0.61 and 2.5 ps (400 nm excitation) and 0.78 and 3.8 ps (460 nm excitation). All DecayAssociatedSpectra(DAS) showed bleachingand absorption. In addition, the first and secondDAS exhibited stimulated emission. Conclusions: The first intermediate state of the PYP photocycleis formed in -0.6 ps and hasa lifetime of -3 ps. Despite the different chromophores, obtained lifetimes strikingly resemblethoseof early intermediatesof bacteriorhodopsin,appearing due to chromophore photoisomerization.
The time resolution (-3 ps) presentlyavailable for protein electricresponsesignal (IJERS)of bR is not enough to assignits early components to this scheme. Here we report ultrafast PERSdata on (i) bR mutants with considerablyprolonged lifetime of the excited stateand (ii) bR samplesof artificial retinal pigment with blocked trans-cisisomerization.
P-E3-19 REDUCTION OR11Y. Dept.of PublicHealth.Graduateschoolof Med. KyotoUniversity (Japan) Purpose: Full descriptionof the spectraland redox behaviorof hemeaA (Fe,,) and heme aB(Fee) that deservesmechanisticanalyses. Methods: Cytochromec oxidasepurified from bovine he&t musclewas reducedwith sodium dithionite underanaerobicconditionsin a stoppedflow rapid-scanappqatus. The spectralchanges recordedfor 30 min were subjectedto data analyses by global fitting to derivekinetic parameters. Results: Basedon a reactionmodel consistingof 11 specieswith specificspectralfeaturesthe observed spectralchangeswere reproducedsati&factorily, thusrigorousanalysesof the reactionmechanism being enabled. Conclusions:The redox potential of FeAis higher than that of Fesby 80 mV. This relationshipand the intrinsicabsorptionspectraof Fe,,end Fesin both oxidizedand reducedstatesare unchanged throughout the reductionprocess.The redox potentialsof both Fe,,and Fea are indep&dent of the redoxstatesof CUB . Thus aqy assumptionlike a ‘ncodasdiqiimoUeI’is neededto a&am rather comp?ica&ispectralchanges&ainrd here.
‘Div. of Chemistryand Cl&i&al Enginee&g, CALTEC, Pasad= (USA), ‘Dept. of Bi Univ. of California at San Diego, La Jolla,9 (7J A) Purpose: To investigatethe relation between the geometricand electronic structuresof the binuclearcenter of CuAin qtochrome oxidase and to study the effect of H&II) on the metal center. Methods: S-, X- and Q-band EPR was used combined with sim+tions and CNDO/S calculationson a 109~atomsystem. Results and ca~~cluslaas: The two Cu ions were nearly identical with gx = 1.996, gv= 2.011, g, = 2.187,,IA+15 G, wd = 29 G and /.4d= 28.5 G. Thelm~w@hwas&depcndentinamanuer ttqimbpy in the A-tensor. ?iI%zt$ &FL IS calculations,basedon the X-ray structureof the Paracoccus dmihijrcons ve, yielded a s’ y ocmqied au&ding orbatalE.a~hCu is A*To ndedtooneSand a’-bQuded to&e other. wast%lmdto extrude one of the Co torrs, T saving the other in the reduced state. 162