Proton-Electron Hyperfine Coupling Constants of the Chlorophyll a Cation Radical by ENDOR Spectroscopy

In this paper we describe the assignment of the major coupling constants in monomer chlorophyll a cation free radical by ENDOR spectroscopy. To facilitate chemical manipulation methylpyrochlorophyllide a has been used as a stand-in, and a suite of six selectively deuterated derivatives have been subjected to ENDOR investigation. Details of the synthesis of these compounds are described. To study the effect of structural features on the spin distribution in the free radicals, six additional chlorophyll derivatives have been studied. Five coupling constants have been assigned, which account for about 80% of the observed electron spin resonance line width in the chlorophyll a monomer cation radical. The spin distribution appears to be highly asymmetric. ENDOR] spectroscopy has provided important support2-* for the original ESR interpretationg-' ' that the primary donor of photosynthesis contains two special chlorophylls. Detailed comparisons of in vivo and in vitro ENDOR data have provided an experimental basis for a variety of special pair chlorophyll model^^^^^-*^ as the primary donor in photosynthesis. In all previous ENDOR studies an interpretation of the spin density distribution in terms of a specific molecular structure has not been documented. For conclusions based on the in vivo ENDOR to be the most convincing, the ENDOR spectrum should account for the whole of the unpaired spin density rather than reflect only a small fraction of an electron distribution. Incomplete interpretation is a likely possibility since all sites of spin density are not necessarily observable by ENDOR spec t ro~copy.~~ Thus a molecular basis for assignment of the ENDOR spectra of the chlorophyll cations is important. In this paper we report the detailed analysis of the protonENDOR spectrum of the cation radical of chlorophyll a (l), methylpyrochlorophyllide a (9), and of a series of related magnesium chlorins. These data provide a clear description of specific assignments of the electron-proton hyperfine coupling constants of in vitro chlorophyll a ?r cation free radical and provide firm support for the view that the ENDOR spectrum of the Chb+ free radical accounts essentially for all of the protons on the chlorophyll macrocycle. Comparisons between in vivo and in vitro ENDOR data therefore do in fact appear to be essentially complete. The model compound (9 ) is essentially identical to chlorophyll a insofar as spin distribution in the cation free radicals is concerned, but it offers a much greater variety of possibilities for structural modifications and for the selective introduction of deuterium. The results of the ENDOR spectroscopy on a wide range of selectively deuterated and structurally modified chlorins establish that chlorophyll a a cation free radical has low spin density at the four methine bridges and the nitrogen atoms of the pyrrole rings. Although the results are in general agreement with open shell calculations for chlorins, the chlorophyll a cation radical shows a strongly asymmetric spin distribution that cannot as yet be completely accounted for by current theory. Finally we show experimentally that this asymmetry is the result of ring V of the chlorophyll macrocycle and is not a result of other various functional side groups. Experimental Section Spectra. ENDOR spectra were recorded on a Varian E-700 spectrometer with a Varian large access cavity and at a temperature of 100 K on samples prepared on the vacuum line. Compounds (0.5-1 mg) were first dried by dissolution in benzene, followed by evaporation Journal of the American Chemical Society / 99:5 / March 2, 1977