Dynamics of carbon monoxide photodissociation in Bradyrhizobium japonicum FixL probed by picosecond midinfrared spectroscopy.

The FixL proteins are heme-based bacterial oxygen sensors, distinct from globins in structure and ligand binding properties. To better understand the dynamics of ligand dissociation and binding within the PAS domain fold of FixL, we have carried out picosecond visible pump-midinfrared probe spectroscopy on the isolated PAS domain of FixL from Bradyrhizobium japonicum. We employ the diatomic ligand CO as a probe of the ligand-dissociation pocket dynamics; upon photoexcitation with a visible laser pulse, CO is released and the infrared-active stretch frequency of the CO molecule changes, as it is very sensitive to interactions with the surrounding protein. The infrared absorption difference spectra indicate that the escape of photolyzed CO to solvent is preceded by transient docking within the protein in a manner similar to globins. A small-scale spectral change of the CO molecule on a picosecond time scale is likely due to changes in heme-protein conformation associated with cooling. A larger scale spectral evolution on a nanosecond time scale indicates a structural change in the protein, possibly related to changes in the beta-strands associated with the transition from CO-bound to deoxy in BjFixLH (Key, J.; Srajer, V.; Pahl, R.; Moffat, K. Biochemistry 2007, 46, 4706).

Hydrogen-bond switching through a radical pair mechanism in a flavin-binding photoreceptor.

BLUF (blue light sensing using FAD) domains constitute a recently discovered class of photoreceptor proteins found in bacteria and eukaryotic algae, where they control a range of physiological responses including photosynthesis gene expression, photophobia, and negative phototaxis. Other than in well known photoreceptors such as the rhodopsins and phytochromes, BLUF domains are sensitive to light through an oxidized flavin rather than an isomerizable cofactor. To understand the physicochemical basis of BLUF domain photoactivation, we have applied femtosecond transient absorption spectroscopy to the Slr1694 BLUF domain of Synechocystis PCC6803. We show that photoactivation of BLUF domains proceeds by means of a radical-pair mechanism, driven by electron and proton transfer from the protein to the flavin, resulting in the transient formation of anionic and neutral flavin radical species that finally result in the long-lived signaling state on a 100-ps timescale. A pronounced deuteration effect is observed on the lifetimes of the intermediate radical species, indicating that proton movements underlie their molecular transformations. We propose a photoactivation mechanism that involves a successive rupture of hydrogen bonds between a conserved tyrosine and glutamine by light-induced electron transfer from tyrosine to flavin and between the glutamine and flavin by subsequent protonation at flavin N5. These events allow a reorientation of the conserved glutamine, resulting in a switching of the hydrogen-bond network connecting the chromophore to the protein, followed by radical-pair recombination, which locks the glutamine in place. It is suggested that the redox potential of flavin generally defines the light sensitivity of flavin-binding photoreceptors.

Purification and initial characterization of a putative blue light-regulated phosphodiesterase from Escherichia coli.

The Escherichia coli protein YcgF contains a photosensory flavin adenine dinucleotide (FAD)-binding BLUF domain covalently linked to an EAL domain, which is predicted to have cyclic-di-guanosine monophosphate (GMP) phosphodiesterase activity. We have cloned, overexpressed and purified this protein, which we refer to as blue light-regulated phosphodiesterase (Blrp) for its putative activity. Blrp undergoes a reversible photocycle after exposure to light in which the spectrum of its photostationary state and kinetics of recovery of the dark state are similar to those of the isolated BLUF domain of the AppA protein. Unlike the AppA BLUF domain, the chromophore environment in the context of full-length Blrp is asymmetric, and the protein does not undergo any detectable global changes on exposure to blue light. When overexpressed in E. coli, Blrp copurifies with certain proteins which suggests that it plays a protective role in response to oxidative stress. Predicted proteins from Klebsiella pneumoniae and from a bacterium in the Sargasso Sea are similar to E. coli Blrp in both their BLUF and EAL domains, which suggests that blue light sensing in these bacteria may follow similar pathways.