Impacts of Cys392, Asp393, and ATP on the FAD Binding, Photoreduction, and the Stability of the Radical State of Chlamydomonas reinhardtii Cryptochrome

Abstract

Plant cryptochromes (CRYs) are blue‐light receptors that regulate light‐dependent growth, development, and circadian rhythms. A flavin adenine dinucleotide (FAD) cofactor is bound to the photolyase homology region (PHR) of plant CRYs and can be photoreduced to a neutral radical state under blue light. This photoreaction can trigger subsequent signal transduction. Plant CRYs can also bind an ATP molecule adjacent to FAD in a pocket of the PHR. Chlamydomonas reinhardtii contains a single plant CRY, named Chlamydomonas photolyase homologue 1 (CPH1). In CPH1, Cys392 and Asp393 are located near the FAD cofactor. Here we have shown that replacing Cys392 with Ser has little effect on the properties of CPH1. The C392N mutant, however, showed a faster photoreduction rate than wild‐type CPH1, together with a significantly lower oxidation rate of the neutral radical state. Substituting an Asn residue for Asp393 in CPH1 improved the binding affinity for FAD as well as the stability of the neutral radical, but photoreduction in the case of this mutant was severely inhibited. In the presence of ATP, CPH1 and its mutants exhibited significantly higher binding affinity for FAD and slower oxidation of the neutral radical. These results reveal that the residues at site 392 and the presence of ATP can tune the stability of the neutral radical, that the Asp residue at site 393 is crucial for photoreduction, and that the photoreduction rate is not determined merely by the stability of the neutral radical in CPH1.

Rapid Oxidation Following Photoreduction in the Avian Cryptochrome4 Photocycle

“…We have previously analyzed the cCRY4 photocycle in the presence of 2–10 mM dithiothreitol (DTT), as the reducing agent, as has been investigated in many other studies. ,− In this study, we consider that this experimental condition may not accurately reflect conditions within the cell, because DTT at >0.8 mM is reported to reduce the viability of the cells under some conditions . Therefore, we compared cCRY4 photoreactions using UV–vis spectroscopy in both the presence and absence of DTT (Figure ).…”

“…In the present study, we aim to investigate transient reactions in cCRY4 photoreduction and dark oxidation using millisecond flash photolysis in the presence and absence of dithiothreitol (DTT), a reductant widely used in previous studies. ,− …”

Investigation of the pH-dependence of the oxidation of FAD in VcCRY-1, a member of the cryptochrome-DASH family

“…While a single amino acid substitution may allow some promiscuity in the protein function, the role of the protein in regulation of the FAD cofactor is more complicated. So, while the aspartate to asparagine point mutation proximal to the N(5)-H in the FAD for the plant cryptochrome-1 from Arabidopsis thaliana (AtCRY-1) allowed the protein to display some PL-like DNA repair [23], the polarity of the amino acid one upstream from the N(5) proximal residue also seems to affect FADHstability [30]. This residue is generally more polar in PL and plant CRY and mostly non-polar in animal CRY; it appears that a more polar residue stabilizes the FADH − state.…”

Die Bedeutung des Flavin-Redoxzustands und der C-terminalen Proteindomäne für die Struktur und das Interaktionsverhalten des pflanzenähnlichen Cryptochroms CryP aus Phaeodactylum tricornutum

“…Durch den Ausschluss von Sauerstoff, die Zugabe von ATP oder Messungen in einem zellulären in vivo Umfeld wird die Redoxumgebung verändert und damit die Stabilität des semireduzierten FAD •bzw. FADH • sowohl in pCRYs als auch aCRYs Typ I auf über 1 h erhöht (Berndt et al 2007;Bouly et al 2007;Hoang et al 2008a;Herbel et al 2013;Vaidya et al 2013;Müller et al 2014;El-Esawi et al 2015;Hense et al 2015;Xu et al 2019 al. 1999;Vitaterna et al 1999).…”