Rhodopsin guanylyl cyclases (RGCs) belong to the 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 domain, that holds the catalytic center inactive in the dark. Structural determinants for activation of the rhodopsin moiety eventually leading to catalytic activity are largely unknown. Here, we investigate the mechanistic role of the D283-C259 (DC) pair that is hydrogen bonded via a water molecule as a crucial functional motif in the homodimeric C. anguillulae RGC. Based on a structural model of the DC pair in the retinal binding pocket obtained by MD simulation, we analyzed formation and kinetics of early and late photocycle intermediates of the rhodopsin domain wild type and specific DC pair mutants by combined UV–Vis and FTIR spectroscopy at ambient and cryo-temperatures. By assigning specific infrared bands to S-H vibrations of C259 we are able to show that the DC pair residues are tightly coupled. We show that deprotonation of D283 occurs 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 early cryo-trapped intermediates. We propose a comprehensive molecular model for 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 from the early J state to the active M state and explains the retarding effect of C259 mutants.