Crystal Structures of a Bacteriophytochrome from Stigmatella aurantiaca Reveal Amino Acids that Modulate Photoconversion

Bacteriophytochromes(Bphs) are photoreceptors that are found in photosynthetic and non-photosynthetic bacteria. Bphs have the ability to undergo reversible photoconversion between spectrally distinct red and far-red light absorbing forms, Pr and Pfr, respectively.  Biliverdin (BV), a linear tetrapyrrole, composed of A-D rings, initiates the photoconversion in Bphs.  Current structural information is based on 4 different Bph structures each solved in the Pr state. Here we report on the X-ray crystallographic structure of the wild type and mutant Bph from Stigmatella aurantiaca (SaBphP), covalently bound to BV in the Pr state.  SaBphP undergoes limited Pr/Pfr photoconversion and is the only Bph with threonine (T) instead of a conserved histidine (H) in the BV-binding domain of the protein.  In published Bph structures, conserved H stabilizes BV in the Pr state by forming a hydrogen bond with the carbonyl group of D ring.  Using site-directed mutagenesis we replaced T with H in SaBphP.  A purified T289H mutant was analyzed using UV-vis absorption spectroscopy. Interestingly, T289H mutant was more photoactive with respect to wild type SaBphP.  Wild type SaBphP and T289H mutant crystal structures were solved in the Pr state at 2.65Å and 2.4Å, respectively.  Proteins crystallized as monomers per asymmetric unit with general topology similar to other Bphs.  In T289H structure we observe hydrogen bond between H and the carbonyl group of the D ring, while in the wild type structure T does not hydrogen bond to BV. Implications of our findings with respect to Bph photoconversion will be discussed.