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Eur J Biochem. 2004 Mar;271(6):1117-26.

Chromophore selectivity in bacterial phytochromes: dissecting the process of chromophore attachment.

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Max-Planck-Institute for Biochemistry, Martinsried, Germany.


Bacterial phytochromes (Bphs) are ancestors of the well characterized plant photoreceptors. Whereas plant phytochromes perform their photoisomerization exclusively via a covalently bound bilin chromophore, Bphs are variable in their chromophore selection. This is demonstrated in the cyanobacterium Calothrix PCC7601 that expresses two Bphs, CphA and CphB. CphA binds phycocyanobilin (PCB) covalently, whereas CphB, lacking the covalently binding cysteine of the plant phytochromes, carries biliverdin IXalpha (BV) as the chromophore. Our experiments elucidate the different modes of chromophore-protein interaction in CphA and CphB and offer a rationale for their chromophore selectivity. The tight binding of BV by CphB prevents PCB from competing for the binding cavity. Even when the chromophore-binding cysteine has been inserted (CphB-mutant L266C), PCB replaces BV very slowly, indicating the tight, but not irreversible binding of BV. The mutant CphB L266C showed a redox-sensitivity with respect to its PCB binding mode: under reducing conditions, the chromoprotein assembly leads to spectra indicative for a covalent binding, whereas absence of dithiothreitol or its removal prior to assembly causes spectra indicative for noncovalent binding. Regarding the CphB-type Bphs lacking the covalently binding cysteine, our results support the involvement of the succeeding histidine residue in chromophore fixation via a Schiff base-like bond between the bilin A-ring carbonyl and the histidine imidazole group. The assembly process and the stability of the holo-proteins were strongly influenced by the concentration of added imidazole (mimicking the histidine side-chain), making the attachment of the chromophore via the histidine more likely than via another cysteine of the protein.

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