The Functionality of the DC Pair in a Rhodopsin Guanylyl Cyclase from Catenaria anguillulae

•Rhodopsin guanylyl cyclases (RGCs) are members of the just recently discovered class of enzymerhodopsins catalyzing the transition from GTP into the second messenger cGMP, whereas light-regulation of enzyme activity is mediated by a membrane bound microbial rhodopsin (Rh) domain. Mechanistic details of rhodopsin activation that triggers the catalytic process are largely unknown.•The role of the C259-D283 (DC) pair as a crucial functional motif in the homodimeric RGC from C. anguillulae were studied. Formation and kinetics of early and late photocycle intermediates of the rhodopsin domain in the wild type and specific mutants of the DC pair were analyzed by combined UV/Vis and FTIR spectroscopy at ambient and cryo-temperatures. Furthermore, the influence of these mutations on the light-induced cGMP production were studied.•The DC pair residues are tightly coupled and deprotonation of D283 occur already in the inactive L-state as a prerequisite for M-state formation, whereas structural changes of C259 occur in the active M-state and the early cryo-trapped intermediates.•We propose a comprehensive model for the formation of the M-state that activates the catalytic moiety. It involves light induced changes in bond strength and hydrogen bonding of the DC pair residues D283 and C259 from the early J state to the active M-state and explains the retarding effect of C259 mutants on a molecular level.