The transitory complex between photoexcited rhodopsin and transducin

Abstract

In the first step of the visual transduction cascade a photoexcited rhodopsin molecule, R ret *, binds to a GDP‐carrying transducin molecule, T GDP . The R*‐T interaction causes the opening of the nucleotide site in T and catalyzes the GDP/GTP exchange by allowing the release of the GDP. We have studied the influences on this R*‐T transitory complex of the occupancies of the nucleotide site in T and the retinal site in rhodopsin. After elimination of the GDP released from the bound transducin, the complex, named R ret *‐T e (ret for retinal present, e for nucleotide site empty) remains stabilized almost indefinitely in a medium whose ionic composition is close to physiological. In this complex the bound T e retains a lasting ability to interact with GDP or GTP, and R ret * remains spectroscopically in the meta‐II state, by contrast with free R ret * which decays to opsin and free retinal. Hence the R*‐T interaction which opens the nucleotide site in T conversely blocks the retinal site in R ret *. Upon prolonged incubation in a low‐ionic‐strength medium the R ret *‐T e complex dissociates partially, but the liberated T e is then unable to rebind GDP or GTP, even in the presence of R ret *; it is probably denaturated. Upon treatment of the R ret *‐T e complex by a high concentration of hydroxylamine, the retinal can be removed from the rhodopsin. The R e ‐T e complex remains stable and the complexed transducin keeps its capacity to bind GTP. T GTP then dissociates from R e . The liberated R e loses its capacity to interact with a new transducin. These data are integrated into a discussion of the development of the cascade. We stress that affinities, i.e. dissociation equilibrium constants, are insufficient to describe the flow of reactions triggered by one R ret * molecule. It depends on a few critical rapid binding and dissociation processes, and is practically insensitive to other slow ones, hence to the values of affinities that express only the ratio of kinetics constants. The effect of the R*‐T interaction on the retinal site in rhodopsin is analogous to the effect of the binding of a G‐protein on the apparent affinity of a receptor for its agonist.