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

Abstract

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...

Electron spin polarization of photosynthetic reactants

“…For the cations the AT-hyperfine splittings are much less, e.g. BChla + : A Ni = -3-2, ^4 Ns = -31, A^a = -2-4 and N 4 = ±°'4 MHz (Hoff & Mobius, 1978;Mobius, Plato & Lubitz, 1982).…”

Biomolecular EPR Meets NMR at High Magnetic Fields

“…The advantages of TRIPLE over ENDOR-enhanced sensitivity and resolution, information about multiplicity and relative signs of hyperfine couplings from line intensity variations-often justify the additional experimental effort inherent to triple resonance spectroscopy. This technique turned out to be extremely powerful in elucidating the electronic structures of organic radicals in solution [154] and transient cofactor radical ion intermediates in the reaction center protein complex of primary photosynthesis [165][166][167][168]. For a detailed discussion of the thorny way of liquid-phase ENDOR to non-proton nuclei, see [152][153][154] where also the original references are given [151][152][153]155,[169][170][171][172][173][174][175][176].…”

Jim Hyde and the ENDOR Connection: A Personal Account

“…Finally, most of the hyperfine couplings (including their signs) were measured. The results were jointly published in PNAS in May 1978, they had been communicated by George Feher in February 1978 [ 49 ]. It is very sad that Arnold Hoff died so early, in 2002 at the age of 63.…”