Unreliability of carotenoid electrochromism for the measure of electrical potential differences induced by ATP hydrolysis in bacterial chromatophores

Abstract

electric potential difference is usually obtained by generating Abstract ATP hydrolysis induces the activation of the proton K+-diffusion potentials in chromatophores treated with valinoATPase in chromatophores of Rhodobacter capsulatus supplemycin [4,10], a procedure normally yielding a linear dependence mented with nigericine and 50 mM K + (i.e. when ApH < 0.2 of the absorption change on the Nernst's potential. It is asunits). The value of transmembrane electric potential (zl~p) drivsumed that the electrochromic signals generated by diffusion ing this activation was measured using three different approaches: carotenoid electrochromism, uptake of SCN-and responses of potentials or by the electrogenic reactions in chromatophores the dye oxonol VI. The value of zt~ calculated from the SCNessentially reflect a potential difference between an inner and uptake, on the basis of an internal volume determined experimenan outer phase isopotential throughout their volumes and, tally, was about 140 mV, while that indicated by the electrotherefore, independent on the nature of the electrogenic reacchromic signal ranged between 35 and 70 inV. Only the value tion causing it. This view neglects any electrostatic phenomeindicated by SCN-distribution is consistent with the energetic non at the interfaces and any possible dishomogeneity along the requirement for the activation of H+-ATPase.membrane plane. It is on this basis that the use of electrochro- [6,7].The large electrochromic signal observed in bacterial chro-2. Materials and methods matophores is due to the carotenoids present in the LH II antenna complex, and corresponds to a red shift of the absorpRhodobacter capsulatus strain GA was grown photoheterotrophically tion spectrum. The extent of the shift, and consequently of the at high light intensity, harvested after 16 hours of growth and chromaamplitude of the absorption changes measured at appropriate tophores were prepared and stored at -18°C as described in [13]. Due to the interference of CI ions with the SCN reversible electrode, in all wavelengths, is proportional, within a wide range, to A~p. This buffers used chloride salts were substituted with the corresponding linear response is thought to be due to pigment molecules elecacetates. trically polarized when associated to the LH II apoprotein [8,9].ATPase activity was measured with a glass electrode (Schott ThaCalibration of the electrochromic optical signal in terms of lamid Model 9218/7.0/300 M.Q) in an apparatus similar to that described in [14] and, occasionally, colorimetrically [13]. SCN-uptake was measured potentiometrically with a SCN -sensi-*Corresponding author. Fax: (39) (51) 242576.rive electrode (Orion, Model 94-58) mounted on the same apparatus used for the ATPase assay. Actinic illumination, provided through the Abbreviations: ain,out , activities in the internal, external compartments; bottom of the measuring cuvette and filtered through 1 cm of water and Bchl, bacteriochlorophyll; H÷-ATPase, ATP-synthase (EC3.6.1.34); a Kodak Wratten 88A gela...

Sulfite Stimulates the ATP Hydrolysis Activity of but not Proton Translocation by the ATP Synthase of Rhodobacter Capsulatus and Interferes with its Activation by ΔH+

“…This mixture includes a number of formal buffers simulating the buffering power of the membrane structural components and the water-soluble buffers contained in the internal lumen of chrornatophores. The internal volume of chromatophores u,, was measured with a EPR probe and corresponded to 15 pl/pmol bacteriochlorophyll (Crimi et al, 1995).…”