Abstract
The choanoflagellate Salpingoeca rosetta contains a chimeric rhodopsin protein composed of an N-terminal rhodopsin (Rh) domain and a C-terminal cyclic nucleotide phosphodiesterase (PDE) domain. The Rh-PDE enzyme light-dependently decreases the concentrations of cyclic nucleotides such as cGMP and cAMP. Photoexcitation of purified full-length Rh-PDE yields an "M" intermediate with a deprotonated Schiff base, and its recovery is much faster than that of the enzyme domain. To gain structural and mechanistic insights into the Rh domain, here we expressed and purified the transmembrane domain of Rh-PDE, Rh-PDE(TMD), and analyzed it with transient absorption, light-induced difference UV-visible, and FTIR spectroscopy methods. These analyses revealed that the "K" intermediate forms within 0.005 ms and converts into the M intermediate with a time constant of 4 ms, with the latter returning to the original state within 4 s. FTIR spectroscopy revealed that all-trans to 13-cis photoisomerization occurs as the primary event during which chromophore distortion is located at the middle of the polyene chain, allowing the Schiff base to form a stronger hydrogen bond. We also noted that the peptide backbone of the α-helix becomes deformed upon M intermediate formation. Results from site-directed mutagenesis suggested that Glu-164 is protonated and that Asp-292 acts as the only Schiff base counterion in Rh-PDE. A strong reduction of enzymatic activity in a D292N variant, but not in an E164Q variant, indicated an important catalytic role of the negative charge at Asp-292. Our findings provide further mechanistic insights into rhodopsin-mediated, light-dependent regulation of second-messenger levels in eukaryotic microbes.
Highlights
The choanoflagellate Salpingoeca rosetta contains a chimeric rhodopsin protein composed of an N-terminal rhodopsin (Rh) domain and a C-terminal cyclic nucleotide phosphodiesterase (PDE) domain
Photoactivated adenylyl cyclase with a FAD chromophore was discovered in Euglena gracilis in 2002 [14], and a microbial rhodopsin containing a guanylyl cyclase (GC) domain was discovered in Blastocladiella emersonii [7]
The present transient absorption study revealed that the K-like intermediate converted into the M intermediate with a time constant of 4 ms and that the M intermediate returned to its original state with a time constant of 4 s
Summary
From a similar broad spectral feature of the bands at 2250 –2100 cmϪ1 (Ϫ) and at 2050 –1900 cmϪ1 (ϩ), it is likely that these bands correspond to each other, presumably the N–D stretch of the Schiff base In this case, a lower frequency shift indicates that the hydrogen bond of the Schiff base becomes stronger in the K intermediate. The spectral changes of amide-I in Rh-PDE(TMD) are from 1658 cmϪ1 in the unphotolyzed state to 1671 cmϪ1 in the M intermediate, indicating that the hydrogen bond of the ␣-helix is disrupted or largely weakened This entire situation is in contrast to the case of. This is the case for CrCCR(C1C2) [20], suggesting the same origin. Two cysteines appear to change the environment of the S–H group, where Fig. 6D shows no deuteration
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