Phytochromes are photochromic photoreceptors with a bilin chromophore that are found in plants and bacteria. The soil bacterium Agrobacterium tumefaciens contains two genes that code for phytochrome-homologous proteins, termed Agrobacterium phytochrome 1 and 2 (Agp1 and Agp2). To analyze its biochemical and spectral properties, Agp1 was purified from the clone of an E. coli overexpressor. The protein was assembled with the chromophores phycocyanobilin and biliverdin, which is the putative natural chromophore, to photoactive holoprotein species. Like other bacterial phytochromes, Agp1 acts as light-regulated His kinase. The biliverdin adduct of Agp1 represents a previously uncharacterized type of phytochrome photoreceptor, because photoreversion from the far-red absorbing form to the red-absorbing form is very inefficient, a feature that is combined with a rapid dark reversion. Biliverdin bound covalently to the protein; blocking experiments and site-directed mutagenesis identified a Cys at position 20 as the binding site. This particular position is outside the region where plant and some cyanobacterial phytochromes attach their chromophore and thus represents a previously uncharacterized binding site. Sequence comparisons imply that the region around Cys-20 is a ring D binding motif in phytochromes.

The Pr --> Pfr phototransformation of the bacteriophytochrome Agp1 from Agrobacterium tumefaciens and the structures of the biliverdin chromophore in the parent states and the cryogenically trapped intermediate Meta-R(C) were investigated with resonance Raman spectroscopy and flash photolysis. Strong similarities with the resonance Raman spectra of plant phytochrome A indicate that in Agp1 the methine bridge isomerization state of the chromophore is ZZZasa in Pr and ZZEssa in Pfr, with all pyrrole nitrogens being protonated. Photoexcitation of Pr is followed by (at least) three thermal relaxation components in the formation of Pfr with time constants of 230 micros and 3.1 and 260 ms. H2O/D2O exchange reveals kinetic isotope effects of 1.9, 2.6, and 1.3 for the respective transitions that are accompanied by changes of the amplitudes. The second and the third relaxation correspond to the formation and decay of Meta-R(C), respectively. Resonance Raman measurements of Meta-R(C) indicate that the chromophore adopts a deprotonated ZZE configuration. Measurements with a pH indicator dye show that formation and decay of Meta-R(C) are associated with proton release and uptake, respectively. The stoichiometry of the proton release corresponds to one proton per photoconverted molecule. The coupling of transient chromophore deprotonation and proton release, which is likely to be an essential element in the Pr --> Pfr photocon-version mechanism of phytochromes in general, may play a crucial role for the structural changes in the final step of the Pfr formation that switch between the active and the inactive state of the photoreceptor.

The complete sequence of the Synechocystis chromosome has revealed a phytochrome-like sequence that yielded an authentic phytochrome when overexpressed in Escherichia coli . In this paper we describe this recombinant Synechocystis phytochrome in more detail. Islands of strong similarity to plant phytochromes were found throughout the cyanobacterial sequence whereas C-terminal homologies identify it as a likely sensory histidine kinase, a family to which plant phytochromes are related. An ≈ 300 residue portion that is important for plant phytochrome function is missing from the Synechocystis sequence, immediately in front of the putative kinase region. The recombinant apoprotein is soluble and can easily be purified to homogeneity by affinity chromatography. Phycocyanobilin and similar tetrapyrroles are covalently attached within seconds, an autocatalytic process followed by slow conformational changes culminating in red-absorbing phytochrome formation. Spectral absorbance characteristics are remarkably similar to those of plant phytochromes, although the conformation of the chromophore is likely to be more helical in the Synechocystis phytochrome. According to size-exclusion chromatography the native recombinant apoproteins and holoproteins elute predominantly as 115- and 170-kDa species, respectively. Both tend to form dimers in vitro and aggregate under low salt conditions. Nevertheless, the purity and solubility of the recombinant gene product make it a most attractive model for molecular studies of phytochrome, including x-ray crystallography.