Flash spectroscopic studies of the kinetics of the halorhodopsin photocycle

Abstract

The photoreactions of halorhodopsin are complicated by the fact that the parent pigment and its photoproducts interact with chloride. Thus, in any photoreaction scheme at least four species have to be accounted for: HR565 and HR578 Cl-, as well as HR640 and HR520 Cl-. A photocycle scheme proposed earlier places the two main photointermediates of halorhodopsin, HR520 Cl- and HR640, into a single photocycle, with a chloride-dependent equilibrium between them [Oesterhelt, D., Hegemann, P., & Tittor, J. (1985) EMBO J. 4, 2351-2356]. This scheme, with the additional feature of direct photoproduction of HR640 from HR565, was tested in this work by using numerical solutions of the appropriate differential equations to simulate flash-induced absorption changes at 500 nm (production of HR520 Cl-) and at 660 nm (production of HR640). The time scale of the simulation was ms following the flash. Comparison of the simulated curves with experimental traces yielded a unique set of three rate constants. The proposed photocycle scheme and these rate constants predict well the shapes and amplitudes of flash traces at various chloride concentrations. It appears from the photocycle scheme, and the numerical values of rate constants, that chloride is bound with high affinity to the parent halorhodopsin molecule, but with much lower affinity to its main photointermediate. This may be the consequence of the fact that in the parent halorhodopsin in the retinal configuration is all-trans, but in the two photointermediates it is 13-cis.

Chemical reconstitution of a chloride pump inactivated by a single point mutation.

“…After addition of guanidinium at a concentration of 0.2 M, a blue-shifted intermediate is detected (Figure 3B) which corresponds to the HR520 intermediate of the wild type photocycle in the presence of chloride or bromide. Therefore the guanidinium-induced change in the photochemical behaviour of HR-R108Q agrees with the change of the wild type photocycle upon binding of anions to site II (Lanyi and Vodyanoy, 1986;Tittor et al, 1987). Guanidinium titration of the HR520 formation measured as maximal absorption change at 490 nm revealed an apparent guanidinium affinity of 8 ±-2 mM, in agreement with the guanidinium affinities measured for the induced transport activity and the absorption shift (Table III).…”

“…Only the main features of the complex pattern of these difference spectra will be considered. A red-shifted intermediate relative to the initial state is transiently detectable, similar to the truncated photocycle of HR wild type in the absence of transportable anions (Lanyi and Vodyanoy, 1986;Tittor et al, 1987). After addition of guanidinium at a concentration of 0.2 M, a blue-shifted intermediate is detected (Figure 3B) which corresponds to the HR520 intermediate of the wild type photocycle in the presence of chloride or bromide.…”

Chloride binding proteins: mechanistic implications for the oxygen-evolving complex of Photosystem II

“…This site is near, but not on, the retinal Schiff base chromophore (Steiner et al 1984, Maeda et al 1985. Chloride binding to this site raises the pKa of the Schiff base by about 2 units (Steiner et al 1984) and stabilizes the 580 nm intermediate in the photocycle (Steiner et al 1984, Lanyi andVodyanoy 1986). Both the binding constant of the anion and the observed ApKa of the Schiff base decrease sharply as the Stokes radius of the anion increases .…”

A defective proton pump, point-mutated bacteriorhodopsin Asp96----Asn is fully reactivated by azide.

“…HR also undergoes trans to cisisomerization but no deprotonation of its Schiff base in the ms time range of the catalytic cycle was found . Instead changes of chloride affinity occur (Lanyi and Vodyanoy, 1986). Occasionally, however, a proton is lost to the aqueous phase and the molecule is trapped in a blue-light-absorbing intermediate state before it is protonated in the time range of seconds and returns to its initial state.…”