Mechanism of Cph1 Phytochrome Assembly from Stopped-Flow Kinetics and Circular Dichroism†

The kinetics and mechanism of the autocatalytic assembly of holo-Cph1 phytochrome (from Synechocystis) from the apoprotein and the bilin chromophores phycocyanobilin (PCB) and phycoerythrobilin (PEB) were investigated by stopped flow and circular dichroism. At 1:1 stoichiometry, pH 7.9, and 10 °C, SVD analysis of the kinetic data for PCB revealed three spectral components involving three transitions with time constants τ1 ∼ 150 ms, τ2 ∼ 2.5 s, and τ3 ∼ 50 s. τ1 was associated with a major red shift and transfer of oscillator strength from the Soret region to the 680 nm region. When the sulfhydryl group of cysteine 259 was blocked with iodoacetamide, preventing the formation of a covalent adduct, a noncovalent red-shifted complex (680 nm) was formed with a time constant of 200 ms. τ1 could thus be assigned to the formation of a noncovalent complex. The absorption changes during τ1 are due to the formation of the extended conformation of the linear tetrapyrrole and to its protonation in the binding pocket. ...