The Cation Radicals of Free Base and Zinc Bacteriochlorin, Bacteriochlorophyll, and Bacteriopheophytin

264

Analysis of the spectrum suggests that the formation of pF involves electron transfer from one bacteriochlorophyll molecule to another within the reaction center, or possibly from bacteriochlorophyll to the bacteriopheophytin of the complex. The initial absorbance changes after flash excitation also include a bleaching of an absorption band at 800 nm. The bleaching decays with T AZ 30 psec. The bleaching appears not to be a secondary effect, but rather to reveal another early step in the primary photochemical reaction.The primary photochemical reaction of bacterial photosynthesis is the transfer of an electron from a bacteriochlorophyll complex, P, to an acceptor, X, whose identity is uncertain (1-3). The electron transfer reaction occurs with a quantum yield of essentially 100% (4), and it occurs with great speed, even at temperatures below 40K. It can be studied in preparations of isolated "reaction centers," which contain three different polypeptides, four equivalents of bacteriochlorophyll, two of bacteriopheophytin, and one each of ubiquinone and nonheme iron (1-3).In a search for clues to the mechanism of the electron transfer reaction, Parson et al. (5) State pF is unlikely to be the lowest excited singlet state of the bacteriochlorophyll complex, P*, because measurements of the fluorescence yield indicate that the lifetime of P* is only 20 to 40 psec when X is in the reduced form (and less when X is not reduced) (6, 7). The proposal that the primary reaction involves an intermediate state such as pF, rather than proceeding directly from P*, would explain a longstanding anomaly in the quantitative relationship between the quantum yields of photochemistry and fluorescence (3-5). The possibility has remained, however, that pF is a side-product which forms only if the normal reaction is blocked. A decision on the role of state pF in photosynthesis, therefore, requires information on whether pF is formed under conditions that permit the electron transfer reaction to occur. We report here on an investigation of this point, using picosecond kinetic techniques. The results appear to establish pF as an intermediate in the electron transfer reaction. While this work was in progress, Kaufmann et al. (8) reached the same conclusion, using somewhat different techniques of psec spectroscopy. MATERIALS AND METHODSReaction centers were obtained from cells of Rhodopseudomonas sphaeroides strain R-26 by the method of Clayton and Wang (9). The detergent lauryldimethylamine oxide was replaced by Triton X-100 by dialysis (5, 10).The apparatus used in the psec experiments was essentially that of Magde and Windsor (11). We shall summarize the approach only briefly here for clarity; ref. 11 provides additional details. A single pulse from a mode-locked Nd+3/glass laser was frequency-doubled to provide a 530 nm excitation flash lasting about 8 psec. This illuminated only a small band across the center of the sample. A measuring flash of white light, which was generated by self phase modulation of residual 1060 nm light from th...

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Picosecond detection of an intermediate in the photochemical reaction of bacterial photosynthesis.

Rockley¹,

Windsor²,

Cogdell³

et al. 1975

264

“…In a crystal this would be the unavoidable impurity such as a quinone generated by oxidation, but in the LH-2 it could be the peptide constituents. The bleaching then produces a BChl radical cation which has an optical absorption band near 925 nm (40,41) and could be a trap for excitations. The calculated Förster energy transfer rate is 10 12 s Ϫ1 .…”

Section: Discussionmentioning

confidence: 99%

Fluorescence and photobleaching dynamics of single light-harvesting complexes

Bopp

Jia

et al. 1997

244

239

Single light-harvesting complexes LH-2 from Rhodopseudomonas acidophila were immobilized on various charged surfaces under physiological conditions. Polarized light experiments showed that the complexes were situated on the surface as nearly upright cylinders. Their f luorescence lifetimes and photobleaching properties were obtained by using a confocal f luorescence microscope with picosecond time resolution. Initially all molecules f luoresced with a lifetime of 1 ؎ 0.2 ns, similar to the bulk value. The photobleaching of one bacteriochlorophyll molecule from the 18-member assembly caused the f luorescence to switch off completely, because of trapping of the mobile excitations by energy transfer. This process was linear in light intensity. On continued irradiation the f luorescence often reappeared, but all molecules did not show the same behavior. Some LH-2 complexes displayed a variation of their quantum yields that was attributed to photoinduced confinement of the excited states and thereby a diminution of the superradiance. Others showed much shorter lifetimes caused by excitation energy traps that are only Ϸ3% efficient. On repeated excitation some molecules entered a noisy state where the f luorescence switched on and off with a correlation time of Ϸ0.1 s. About 490 molecules were examined.

“…We conclude that the lines are genuine ENDOR transitions. They cannot be attributed to nuclei having a free precession 13 14 Triple resonance In Fig. 2 the high-frequency part of the proton ENDOR spectrum is displayed, together with a Triple resonance spectrum (25,26) indicates that the largest of these splittings is due to the methyl on ring III.…”

Section: Nitrogen Endormentioning

confidence: 99%

“…By comparing the linewidth of the BChl+ electron spin resonance (ESR) signal at X and Q bands for fully protonated and fully'deuterated BChl, McElroy et al (12) estimated a value of 1.2 G for the average nitrogen hyperfine coupling aN. Fajer et al (13)(14)(15) (17). Knowledge of the detailed…”

mentioning

confidence: 99%

Nitrogen electron nuclear double resonance and proton triple resonance experiments on the bacteriochlorophyll cation in solution.

Hoff¹,

Möbius²

1978

Electron nuclear double resonance signals of the pyrrole nitrogens of bacteriochlorophyll a cation in a solution of CH2CI2/CH3OH (6:1) have been observed. The nitrogens are inequivalent: the hyperfine coupling constants were determined to be 2.36 and 3.18 MHz (0.84 and 1.14 C, respectively In recent years much has been learned about the identity of the primary reactants in bacterial photosynthesis. It has been demonstrated (1-3) that the primary electron donor is a pair of bacteriochlorophyll (BChl) molecules, possibly linked by two water molecules (4-7), that the first acceptor is most probably a bacteriopheophytin (8-10), and that the secondary acceptor (formerly called primary acceptor) is an iron-ubiquinone complex (11). It is clearly of prime importance to know the detailed electronic structure of the complex of reactants in order to understand the process of charge separation, which is the basis of photosynthesis. Electron nuclear double resonance (ENDOR) experiments at low temperatures (1-3) gave information about some of the hyperfine coupling constants of the cation of the in vivo primary donor. Similarly, liquid-state ENDOR experiments yielded seven of the proton hyperfine couplings of the monomeric BChl+ cation in organic solution. By inference, one may then calculate the corresponding spin densities for the dimeric primary donor. Conspicuously lacking, however, are direct data on the hyperfine coupling at the site of the four pyrrole nitrogen atoms. By comparing the linewidth of the BChl+ electron spin resonance (ESR) signal at X and Q bands for fully protonated and fully'deuterated BChl, McElroy et al. (12) electronic structure of the binding of Mg to the four pyrrole nitrogens is essential to our understanding of the role of Mg in the primary act of photosynthesis. Knowledge of the spin density on the nitrogens of the cation is an aid to such understanding and is important for use in refinements of quantum chemical calculations.In this communication we report accurate values of the hyperfine coupling of the pyrrole nitrogen in the BChl+ a molecule. We found that the N atoms are inequivalent. The measured hyperfine coupling values agree well with previous estimates (12,13). We also report on Triple proton ENDOR experiments. Accurate values and signs are given for the hyperfine couplings of the two methyl groups, the five ,B protons, and the three methine protons that have measurable hyperfine interaction with the unpaired electron. These data are a complement to, and a refinement of, the data presented by Borg et al. (18) in a recent communication. EXPERIMENTALBacteriochlorophyll a (BChl) was prepared from Rhodospirilium rubrum as described (12). No contaminants were present, as checked by thin-layer chromatography and spectral analysis. From a stock solution of 0.7 mM in dimethylether, aliquots were taken and dried under reduced pressure of 50 nm Hg. Iodine was purified by sublimation and dissolved in CH2Cl2/CH3OH (6:1) to a concentration of 2 mM. Three hundred microliters of the iodine so...