Unusual kinetics of thermal decay of dim-light photoreceptors in vertebrate vision

Abstract

We present measurements of rate constants for thermal-induced reactions of the 11-cis retinyl chromophore in vertebrate visual pigment rhodopsin, a process that produces noise and limits the sensitivity of vision in dim light. At temperatures of 52.0-64.6°C, the rate constants fit well to an Arrhenius straight line with, however, an unexpectedly large activation energy of 114 ± 8 kcal/mol, which is much larger than the 60-kcal/mol photoactivation energy at 500 nm. Moreover, we obtain an unprecedentedly large prefactor of 10 72±5 s −1 , which is roughly 60 orders of magnitude larger than typical frequencies of molecular motions! At lower temperatures, the measured Arrhenius parameters become more normal: E a = 22 ± 2 kcal/mol and A pref = 10 9±1 s −1 in the range of 37.0-44.5°C. We present a theoretical framework and supporting calculations that attribute this unusual temperature-dependent kinetics of rhodopsin to a lowering of the reaction barrier at higher temperatures due to entropy-driven partial breakup of the rigid hydrogen-bonding network that hinders the reaction at lower temperatures.non-Arrhenius | dim-light vision | transition state theory | isomerization rate R hodopsin is a vertebrate dim-light photoreceptor. Molecular studies of rhodopsin in recent decades have largely focused on its photochemistry and photoactivation (1-3). However, complete understanding of rhodopsin's function requires characterization of its thermal properties because thermal isomerization of the 11-cis retinyl chromophore can trigger the same physiological response as photo-isomerization, generating false visual signals as dark noise that jeopardizes photosensitivity (4-6). To enhance dim-light vision, rhodopsin has evolved to acquire remarkable thermal stability with a half-life of 420 y as determined by electrophysiological experiments using the outer segments of rod cells at 36°C (4). However, the molecular mechanism for the thermal stability has remained unclear. Here, we have addressed this by exploring the temperature dependence of thermal decay rate constants k(T) associated with isomerization of the retinyl chromophore and hydrolysis of the chromophore protonated Schiff-base (PSB) linkage, for temperatures ranging from the physiological temperature of 37.0°C to 64.6°C. In the upper range of temperatures from 52.0°C to 64.6°C, we find that the rate constants determined by UV-visible spectroscopy follow a linear Arrhenius model, k(T) = A pref exp(−E a /k B T), where k B is the Boltzmann constant (Fig. 1A). The slope, however, is very steep, giving an elevated activation energy, E a = 114 ± 8 kcal/mol. Surprisingly, this value is much higher than the photoactivation energy at visible wavelengths (60 kcal/mol at 500 nm). E a is also much higher than the reaction enthalpy change (32-35 kcal/mol) (7, 8) (Fig. 1B). In the lower temperature range of our rate constant measurements, 37.0-44.5°C, the slope of the Arrhenius plot decreases abruptly, as shown in Fig. 1A. Fitting a straight line through the low temperature points prod...

Photocyclic behavior of rhodopsin induced by an atypical isomerization mechanism

“…The energy threshold for G t activation by Rh6mr was similar to the Rh activation energy of 9.6 kcal/mol (Fig. 1B, black dashes) (20,21). These data suggest a similar mechanism of G t activation for both Rh6mr and Rh that involves opening of the opsin cytoplasmic side upon light illumination (22,23) and subsequent binding to G t .…”

Measurement of Slow Spontaneous Release of 11-cis-Retinal from Rhodopsin

“…We found that the rate of the light-independent chromophore dissociation at 37 C was 2.2 Â 10 À7 s À1 , corresponding to a half-time of 36 days. This dissociation rate and the activation energy are distinct from the earlier biochemical data that measured the denaturation rate of Rho and opsin in vitro (8)(9)(10), and from the electrophysiological studies that measured thermal activation of single Rho molecules in native disc membranes (4,5). We conclude that the direct breaking of the PSB bond gave rise to the observed chromophore release.…”

“…However, this value was derived from a single temperature, which makes comparison with other thermally activated processes difficult, and the experiments could not definitely distinguish between de novo pigment synthesis and chromophore exchange within the existing pigment. Moreover, all these in vivo data are at odds with the in vitro biochemical measurements, which found that the thermal decay rates of Rho at physiological temperature is on the order of 10 À6 s À1 (8,9). In our study, we revisited the stability of dark-state Rho by measuring the exchange rate of bound 11CR with free 9-cis-retinal (9CR) in Rho reconstituted into detergent/lipid bicelles.…”

“…5 and S1; Table S2). Janz and Farrens (8) and Guo et al (9) described the measurement of the thermal decay of Rho in DM micelles using the decrease of the 500-nm peak as the read-out. We noticed that Janz and Farrens performed their measurement at 1-min interval over a time course of hours (8).…”

“…We noticed that Janz and Farrens performed their measurement at 1-min interval over a time course of hours (8). By comparison, Guo et al derived the decay rates from fewer time points (9). To obtain the absorption spectrum, a small fraction of dark-state Rho was necessarily photobleached.…”