Association between a photo-intermediate of a M-lacking mutant D75N of pharaonis phoborhodopsin and its cognate transducer

Pharaonis phoborhodopsin (ppR or pharaonis sensory rhodopsin II) is a receptor of the negative phototaxis of Natronobacterium pharaonis and forms a complex with its transducer pHtrII in membranes. Flash-photolyis of a D75N mutant did not yield the M-intermediate, but an O-like intermediate is observed in a ms time range. We examined the interaction between the D75N of ppR and t-Htr (truncated pHtrII). These formed a complex in the presence of 0.1% n-dodecyl-β-maltoside, and the association accelerated the decay of the O of D75N from 15 to 56 s^-1. From the decay time constants under varying ratios of D75N and t-Htr, n, the molar ratio of D75N/t-Htr in the complex, and K_D, the dissociation constant, were estimated. The value of n was unity and K_D was estimated to 146 nM. This K_D value can be considered to be the association between the photo-intermediate and t-Htr, which is deduced by the method of estimation. Previously we (Photochem. Photobiol. 74 (2001) 489) reported a K_D of 15 μM for the interaction between the wild-type and t-Htr by means of the change in M-decay rates. Therefore, this value should be the K_D value for the interaction between M of the wild-type and t-Htr.