Radical Formation in the Photoactivated Adenylate Cyclase OaPAC Revealed by Ultrafast Spectroscopy

Abstract

Adenylate cyclases (AC) form a class of enzymes which are involved in the conversion of ATP to cAMP, playing an important role in many regulatory processes as the cAMP produced is involved in signalling pathways controlled by cAMP binding proteins. There is a very special subgroup of AC enzymes called photoactivated adenylate cyclases (PAC) where the ATP to cAMP conversion is controlled by light. Interestingly the photoregulation is controlled by BLUF (Blue light sensing using FAD) domains which are responsible for light regulation in many processes like phototoaxis, photophobia etc. Light regulation of BLUF domains is governed by the reorganization of the hydrogen bond network around the flavin and it is reflected in a ∼10 nm red shift of the flavin absorption peak. The first discovered photo-activated adenylate cyclase was found in a unicellular flagellate Euglenagracilis where the flagellar movement is regulated via a photoactivated AC. Another photoactivated adenylate cyclase called bPAC was discovered in the sulfide-oxidizing bacterium Beggiatoa sp. where the light regulation is solved by the BLUF domain located at the N-terminal part of the protein, the cAMP conversion is accomplished by a class IIIb type adenylyl cyclase at the C-terminal. Another promising PAC was discovered recently in the photosynthetic cyanobacterium Oscillatoriaacuminata which shows substantial activation (20-fold increase in the cAMP level in light compared to the dark) upon light illumination. OaPAC is a 366-aa residue long homodimer having a class III adenylate cyclase domain at the C-terminal and a BLUF domain at the N-terminal. In this work we characterized the primary photochemistry of OaPAC by the means of ultrafast (femtosecond to nanosecond) spectroscopy. Ultrafast transient visible and transient infrared absorption spectroscopy reveals that neutral flavin radical is formed after the blue light absorption.