The interaction of the potential-sensitive molecular probe merocyanine 540 with phosphorylating beef heart submitochondrial particles under equilibrium and time-resolved conditions

Abstract

The interaction of the potential-sensitive extrinsic molecular probe merocyanine 540 (M540) with phosphorylating submitochondrial particles has been investigated under equilibrium and time-resolved conditions. The addition of ATP to a M540-membrane suspension produces oligomycin and CCCP-sensitive spectral changes with absolute maxima near 490, 530, and 565 nm; a 1- to 2-nm red shift of the dye absorption spectrum is also evident in the longer-wavelength region of the spectrum. In fixed-wavelength work, the energy-dependent optical signals were increased by the addition of nigericin and NH 4Cl, and could be subsequently restored to the control level by valinomycin or KSCN, respectively. These observations suggest that M540 is specifically sensitive to the membrane-potential portion of the electrochemical gradient presumably present in the submitochondrial particle system in the presence of substrate. Binding analyses based on the Langmuir adsorption isotherm and the direct linear method indicate that the M540 dissociation constant is decreased by the presence of ATP with little or no change in the maximum number of binding sites. The M540 dissociation constant was markedly decreased when 0.1 m NaCl was present in the medium, suggesting that the association of this probe with the membrane may be subject to considerable surface charge repulsion. Results from the binding analyses indicate that the origin of the energy-dependent spectral changes may be an enhanced association of M540 with the submitochondrial particle membrane resulting from the transfer of dye from the aqueous phase to membrane-binding sites. The time course of the NADH-, ATP-, or succinate-induced signal developed slowly, on a time scale of tens of seconds, and follows a second-order rate law, suggesting that the rate-limiting step in the development of the ATP-induced M540 signal may be the transfer of dye from the aqueous phase to membranebinding sites. The enhanced passive binding of M540 to the submitochondrial particle membrane in the presence of NaCl reduces the concentration of free dye apparently available to redistribute to the membrane when substrate is present, with a concomitant reduction in the observed pseudo-first-order and the second-order rate constants. If the effective free dye concentration is estimated from binding data and used in the plot from which the latter rate constant is obtained, the value of this constant compares favorably with the obtained in the absence of the electrolyte. In a concentration range as large as 0 to 10 μ m, the presence of M540 in submitochondrial particles has no effect on the rate constants characterizing three energy-dependent processes: the ATP-induced cytochrome c oxidase soret band shift, and the oxidation of cytochrome 1 and the reduction of pyridine nucleotide, both via ATP-driven reversed electron transport. The failure of M540 to impede the rates at which these energy-intensive processes develop suggests that the M540 anion does not permeate the particle membrane in the time scale covered by these experiments, 0.3 s to some 15 min.