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

Abstract

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 degrees C, SVD analysis of the kinetic data for PCB revealed three spectral components involving three transitions with time constants tau(1) approximately 150 ms, tau(2) approximately 2.5 s, and tau(3) approximately 50 s. Tau(1) 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. Tau(1) could thus be assigned to the formation of a noncovalent complex. The absorption changes during tau(1) are due to the formation of the extended conformation of the linear tetrapyrrole and to its protonation in the binding pocket. From the concentration and pH dependence of the kinetics we obtained a value of 1.5 microM for the K(D) of this noncovalent complex and a value of 8.4 for the pK(a) of the proton donor. The tau(2) component was associated with a blue shift of about 25 nm and was attributed to the formation of the covalent bond (P(r)), accompanied with the loss of the 3-3' double bond to ring A. Tau(3) was due to photoconversion to P(fr). For PEB, which is not photochromic, the formation of the noncovalent complex is faster (tau(1) = 70 ms), but the covalent bond formation is about 80 times slower (tau(2) = 200 s) than with the natural chromophore PCB. The CD spectra of the PCB adduct in the 250-800 nm range show that the chromophore geometries in P(r) and P(fr) are similar to those in plant phytochrome. The opposite rotational strengths of P(r) and P(fr) in the longest wavelength band suggest that the photoisomerization induces a reversal of the chirality. The Cph1 complex with noncovalently bound PCB was still photochromic when cysteine 259 was blocked with IAA or with the bulkier IAF. The covalent linkage to cysteine 259 is thus not required for photoconversion. The CD spectra of the noncovalently bound PCB in P(r)- and P(fr)-like states are qualitatively similar to those of the covalent adducts, suggesting analogous structures in the binding pocket. The noncovalent interactions with the binding pocket are apparently sufficient to hold the chromophore in the appropriate geometry for photoisomerization.

Eukaryotic algal phytochromes span the visible spectrum

“…5). Similar Soret features are seen in Cph1 and in plant phytochromes (32,45,46); therefore, a similar interpretation would seem likely for those proteins as well. We conclude that significant structural heterogeneity of the bilin configuration itself must exist in the P fr state, even though previous studies demonstrate structural rigidity of the phytochrome P fr state (71) and apparent P fr homogeneity on an ultrafast timescale (72).…”

“…4A). In the P fr state, we observed positive far-red CD and complex Soret features that were similar to those of the model cyanobacterial phytochrome Cph1 (32,46). In surprising contrast, the orange/far-red NpyrPHY1 exhibited negative far-red CD but similar Soret features (Fig.…”

“…The short-wavelength Soret transition also changes in these cases. The simple positive Soret signal seen for P r states is replaced by a complex pattern having an apparent positive band at ∼430 nm, a negative band at ∼380 nm, and another positive band at ∼340 nm (46).…”

“…The two bilin transitions in the range from 250 to 900 nm give opposite CD signs and can provide information about the 3D orientation of the bilin conjugated π system (44). In plant and cyanobacterial phytochromes, the CD of the long-wavelength transition (red-or far-red-absorbing, also known as Q band) inverts upon photoconversion, from negative to positive (32,45,46). The short-wavelength Soret transition also changes in these cases.…”

The Structure of Phytochrome: A Picture Is Worth a Thousand Spectra

“…In this regard, examination of chromophore assembly with Cph1 by stoppedflow spectrophotometry (Borucki et al, 2003) demonstrated that assembly involves three key steps: initial noncovalent binding of PCB to the Cph1 apoprotein in a cyclic, porphyrin-like conformation, formation of a more extended red-shifted PCB Á apoCph1 intermediate, and covalent bond formation concomitant with a spectral blue shift. The red-shifted intermediate is thought to reflect chromophore protonation and adoption of the more extended configuration characteristic of Pr.…”