An Electron-Nuclear Double Resonance (Endor) Study of the Special Pair Model for Photo-Reactive Chlorophyll in Photosynthesis

Abstract

A comparison of hyperfine coupling constants obtained by electron-nuclear double resonance spectroscopy of in vitro monomer chlorophyll and bacteriochlorophyll free radicals with those of the photoesr (electron spin resonance) signal associated with light conversion in photosynthesis provides convincing support for the special pair model for the in vivo photo-reaction center. The special pair model for photo-active chlorophyll predicts that the esr line shape will be narrowed by a factor 1/A/2 relative to monomer Chl at, and that each and every electronnuclear hyperfine (hf) coupling constant in a special pair will be reduced by a factor of 1/2 relative to the monomer coupling constants. A comparison of hf splittings measured in both in vivo and in vitro chlorophyll free radicals is thus a considerably more rigorous test of the special pair proposal. Because chlorophyll free radical esr signals, especially in vivo, do not show hf structures that permit extraction of hf coupling constants, we have had resort to electron-nuclear double resonance (endor) spectroscopy, a high resolution extension of esr first discovered by G. Feher (22), which in combination with organisms and chlorophylls of unnatural isotopic composition makes assignment of the endor spectrum and hf coupling constants possible. MATERIALS AND METHODSInstrumentation. Endor spectra were recorded at 10'K and 108'K on a modified Varian E-700 spectrometer.Production of Chlorophyll Free Radicals. In vitro free radicals were usually generated in 10-4 10-1 M Chl solutions in thoroughly degassed C2H2C12-C2H302H by chemical oxidation with minimal amounts of iodine (23), ferric chloride, or zinc tetraphenylporphyrin free radical (24).In vivo free radicals for endor were generated in packed whole cells, chromatophores, chloroplasts, and reaction center preparations by careful titration with KFe(CN)6. The esr and endor signals produced by this chemical method are indistinguishable from the photo-induced signal in bacteria (16) and green algae and thus are believed to have the same origin.

Energetics of the primary electron transfer reaction revealed by ultrafast spectroscopy on modified bacterial reaction centers

“…The dynamics of this process has been investigated by time-resolved spectroscopy, complementing the structural data. Optical excitation leads to the transfer of an electron from a pair of strongly coupled bacteriochlorophyll molecules [4] to a quinone on the opposite side of the membrane (see inset Fig. 1).…”

Structural studies of the primary donor cation radical P 870 +· in reaction centers of Rhodospirillum rubrum by electron-nuclear double resonance in solution

“…1). Further support for this model came from electron-nuclear double resonance (ENDOR) at low temperatures in which a reduction of the hyperfine coupling constants (hfcs) by a factor of two was deduced when going from BChl-a+ to P8+70 (8)(9)(10)(11)(12)(13)(14). The detailed structure of the suggested dimer is still controversial (3,4,14), but its geometry is of prime importance for basic understanding of the primary act of light-induced charge separation in photosynthesis.…”

“…Liquid-state ENDOR is superior to frozen-solution EN-DOR (10,21,22) because the linewidths are smaller due to the absence of anisotropic broadening so that hfcs from all magnetic nuclei in the radical can often be obtained (23,24). However, most of the ENDOR studies of photosynthesis thus far were carried out in frozen matrices (8)(9)(10)(11)(12)(13)). An EN-DOR in solution study on RC [Rhodopseudomonas (Rp.…”

Ligated chlorophyll cation radicals: Their function in photosystem II of plant photosynthesis

“…ESR data show (25) that the 7 G (1 G = 1o-4 tesla) peak-to-peak linewidth (AH) of P-700+ is narrower by V-than that of Chl+-(AH = 9 G) in dichloromethane and dichloromethane/methanol solutions, as expected if the unpaired electron of the radical is equally shared between two Chl molecules. Additional support for delocalization of spin over two Chls in P-700+-derives from ENDOR spectra of oxidized chloroplasts (34,35) in which several proton hyperfine splittings are found to be approximately half those of Chl+-in vitro (37,38). The nature of P-680+' in PS II is considerably more ambiguous: linewidths of ESR signals attributed to P-680+-range from 7 to 9 G (27-32), resulting in proposals of monomer (28), dimer (29-31), and even trimer (32) configurations.…”

“…Additional support for delocalization of spin over two Chls in P-700+-derives from ENDOR spectra of oxidized chloroplasts (34,35) in which several proton hyperfine splittings are found to be approximately half those of Chl+-in vitro (37,38). The nature of P-680+' in PS II is considerably more ambiguous: linewidths of ESR signals attributed to P-680+-range from 7 to 9 G (27)(28)(29)(30)(31)(32), resulting in proposals of monomer (28), dimer (29)(30)(31), and even trimer (32) configurations.…”