A large absorbance change with similar characteristics had been identified earlier in the photosynthetic bacterium Rhodobacter sphaeroides by Jan Amesz, who had shown that the change could be modelled by a small red shift of the carotenoid absorbance bands. A similar change had been investigated in Rhodospirillum rubrum by Meg Baltscheffsky and Britton Chance. Taking advantage of the lower permeability of chromatophore membranes, and a familiarity with ionophoric mechanisms, Jackson and Crofts were able to demonstrate that the carotenoid electrochromic changes in Rb. sphaeroides were proportional to membrane potential. They used the ionophore valinomycin, which renders membranes specifically permeable to K+-ion, and thus allows membrane potentials of known magnitude to be generated by setting up gradients of K+ (as KCl) before addition of valinomycin.
The electrochromic changes were easier to characterize in the photosynthetic bacteria because the carotenoid absorbance bands were well separated from those of the bacteriochlorophylls. Furthermore, mutant strains were developed that had fewer carotenoids (the G1C strain had Got 1 Carotenoid), which greatly simplified spectral analysis. Work from two competing groups identified the main characteristics of the absorbance change, and let to the recognition of some interesting paradoxes. Holmes and Crofts showed by careful measurment of the shift as a function of amplitude that the shift was very much smaller than expected from a simple electrochromic effect, and started at a point in the spectrum to the red of the bulk carotenoid absorbance. This was especially apparent on integration of the light minus dark difference spectrum (showing the shift), which showed that the spectrum of the shifted component was to the red. Amesz and de Grooth analyzed in great detail the kinetics of changes close to the pseudo-isobest of the difference spectrum, and showed that the change was indeed a shift. The results could be explained by suggesting that the absorbance change was a small shift of the carotenoid spectrum, but that the carotenoid population was not homogeneous. This inhomogeneity must reflect at least two populations, one of which (the active population) was red-shifted with respect to the other.
The frequency shift associated with an electronic transition, Dn (in cm-1), which is experienced by a molecule in an electric field, is given by:
In Rb. sphaeroides GIC, the carotenoid molecule which showed an electrochromic shift was neurosporene, which is known to have no permanent dipole, and yet, as in the wild-type, experiments with valinomycin-induced K+ diffusion potentials showed that the shift was linear with field strength. This could be explained by suggesting that the carotenoids were experiencing a local field from the protein environment of the light-havesting complex, which polarized them, to give an induced dipole. An appropriate expression for the shift would include the polarizing local field, FL, and the vector of the delocalized field in the direction of the induced dipole, FD, which gives rise to the observed linear shift, as separate terms:
The electrochromic changes in Rb. sphaeroides and the closely related Rb. capsulatus have been explored in detail. A fast phase associated with turnover of the photochemical reaction center, and immediate secondary donors and acceptors was resolved into two phases. Phase I was very rapid, and linked to the photochemistry; phase II was in the range 30-100 ms, and was associated with secondary electron transfer. A slower phase (phase III) was associated with turnover of the bc1 complex, as shown by its sensitivity to specific inhibitors such as antimycin and myxothiazol. This later phase has been much studies in the context of the Q-cycle mechanism, since it reflects the electrogenic processes associated with turnover of the complex.
Reactions that have been investigated using this approach are: