Absorption and Emission Spectroscopic Investigation of Thermal Dynamics and Photo- Dynamics of the Rhodopsin Domain of the Rhodopsin-Guanylyl Cyclase from the Aquatic Fungus Blastocladiella emersonii

A new class of rhodopsins covalently linked to an enzymatic domain was discovered recently. A member of this class of enzymerhodopsins, the rhodopsin-guanylyl cyclase (RhGC) was identified in the aquatic fungus Blastocladiella emersonii (BE). Characterization of RhGC showed that the second-messenger molecule cGMP (cyclic guanylyl monophosphate) is produced upon green light illumination. Here, the rhodopsin domain Rh (BE) of the rhodopsin-guanylyl cyclase RhGC was studied by absorption and emission spectroscopic methods. It was found that fresh thawed Rh (BE) was composed of a mixture of retinal – protein conformations. These retinal conformations are likely all-trans protonated retinal Schiff base (Ret_1), 13-cis protonated retinal Schiff base with repositioned counter ion (Ret_2), alltrans protonated retinal Schiff base with repositioned counter ion (Ret_3), and deprotonated all-trans protonated retinal Schiff base (Ret_4). The Rh (BE) thermal denaturing was studied: An apparent protein melting temperature of θm ≈ 49 °C was determined; the apparent protein melting time at room temperature (≈ 21.9 °C) was tm ≈ 1.45 h. Thermal retinal conformation restructuring with irreversible conversion likely to deprotonated 13-cis retinal Schiff base (Ret_4’) was observed. The photo-excitation of all-trans protonated retinal Schiff base (Ret_1) caused a primary photocycle dynamics involving excited-state picosecond all-trans – 13cis isomerization (13-cis protonated retinal Schiff base Ret_5 formation) followed by ground-state sub-second intermediate retinal Schiff base formations (Ret_2, Ret_3, Ret_4) and subsecond to second recovery to the initial all-trans protonated retinal Schiff base (Ret_1). Long-time all-trans protonated retinal Schiff base photo-excitation caused irreversible (likely 13-cis) retinal Schiff base (Ret_4’) formation.