J. CHEM. SOC. PERKIN TRANS. I 1983 Co-ordination and Aggregation of Bacteriochlorophyll a : An N.M.R. and Electronic Absorption Study Richard G. Brereton and Jeremy K. M. Sanders University Chemical Laboratory, Lensfield Road, Cambridge CB2 I EW ~~~~~~~ ~~ ~ A detailed analysis is presented of the aggregation and co-ordination equilibria available to bacteriochloro- phyll a, and n.m.r. spectroscopic titration experiments are proposed for their investigation. The results show that bacteriochlorophyll a is dimeric in wet benzene, five-co-ordinate monomeric in acetone or ether solution with one bound solvent molecule, and six-co-ordinate monomeric in pyridine or methanol solution with two bound solvent molecules. The second derivatives of the electronic absorption spectra show separate resolved peaks for five- and six-co-ordinate species near 575 and 600 nm respectively.The solution equilibria of chlorophylls are dominated by the co-ordination demands of the central Mg2+ ion; it seems likely that co-ordination state also plays an important role in photosynthesis.1+2It is well established that Mg prefers to be five- or six-fold c~-ordinated,~-~ but only four sites are occupied by the pyrrole nitrogens in chlorophylls. The resulting co- ordinative unsaturation must be satisfied by a fifth and, in some circumstances, sixth basic axial ligand (Figure 1). In the absence of an external ligand the vacant co-ordination site is satisfied by a further chlorophyll molecule; this second mole- cule either co-ordinates directly using a carbonyl group or indirectly via a water molecule which hydrogen bonds to the carb~nyl.~-~Hence chlorophylls exhibit a tendency to self aggregate and so possess correspondingly complicated solu- tion equilibria.Although the aggregation and co-ordination of chlorophyll a (Chl a) have been much studied by i.r. spectroscopy, solution osmometry and 'H n.m.r. spectroscopy, they are still a matter of debate;4 by contrast, because both the availability and stability of bacteriochlorophyll a (BChl) (1) are generally believed to be low, less work has been done on BChl and the effect on its chemistry is even less clear. Here we report spectroscopic studies which elucidate BChl co-ordination properties in a variety of solvents.The follow- ing paper reports evidence that the solution and solid-state L L I I i Figure 1. Schematic diagrams of bacteriochlorophyll a when five- co-ordinate (left) and six-co-ordinate (right) stability of BChl are in part dependent on the co-ordination state and describes how we have used this evidence to design an improved high-yielding extraction procedure which gives stable microcrystalline BChl. In the third paper 'we exploit this knowledge about the stability of BChl to provide con- centrated but stable solutions suitable for natural abundance I3C n.m.r. spectroscopy. A related paper will describe in detail the methods used for determining the equilibrium constants in this paper.* There is a strong body of evidence to suggest that the molecules of BChl associate together in the photosynthetic reaction centre in vivo and that this complex is the primary electron donor of photosynthesi~.~~~~N.m.r.shift titrations on isolated BChl suggested that small ligands bind onto and dis- sociate BChl aggregates. Electronic absorption spectra indicate the monomeric BChl in solution can be either five- or six-fold co-ordinated :Evans and Katz suggest that the pos- ition of the Qx band is strongly dependent on the co-ordination state, absorbing at ca. 580 nm for five-co-ordinate and 600 nm for six-co-ordinate material." We extend these two observ- ations to build a more detailed picture, but present first a description of the solution equilibria of BChl and a summary of the theory of the experimental design.Principles.-The Scheme illustrates the principal BChl solution equilibria. We define the following constants : where KOis the constant for dissociation of BChl, aggregates to BChl, dimers, BChl*LI2K--BChl,.L2 where Kl is the disaggregation constant of a dimer induced by ligand L, where K2is the ligand binding constant to monomeric BChl, where K3 is the dimerisation constant for BChl and K-BChl*LJ -BChl-LL (51 424 J. CHEM. SOC. PERKIN TRANS. I 1983 BChl BChl, 11. BChl.L BChl.L, Scheme where K4 is the second ligand binding constant. These are convenient definitions, although strictly KJ to K4are not all independent variables. We can induce the equilibria in the Scheme by changing the concentrations of L or BChl.The equilibria actually observed depend on the relative sizes of the various constants. The total concentration of ligand needs to be considerably in excess of that of BChl for significant disaggregation shifts to be observable. Hence we monitored the BChl chemical shifts whilst varying the concentration of L, assuming fast exchange between species of different chemical shifts. For easy interpretation of such shifts it was necessary to ensure that the only effective process occurring during ligand titration was of the form BChl, + rn . n . L +n(BChl . L,,,).Conditions for this to be the case were established as follows. (a) For the BChl to be initially more than 90 in the dimer form it was necessary for (1 + 8K3c)i > 19, where c is the total BChl concentration.8 Assuming that for BChl, K3 is similar to that for chlorophyll a (ca.lo4mol 1-I) then this im- poses a lower limit on c of ca. 4.5 mM. Experimentally we con- firmed that BChl shifts were Concentration independent in the 5-15 mM range used in this work. (b) For ease of analysis it was convenient to assume only a small proportion (展开▼
