Distinct classes of red/far-red photochemistry within the phytochrome superfamily

Abstract

Phytochromes are a widespread family of photosensory proteins first discovered in plants, which measure the ratio of red to far-red light to control many aspects of growth and development. Phytochromes interconvert between red-absorbing P(r) and far-red-absorbing P(fr) states via photoisomerization of a covalently-bound linear tetrapyrrole (bilin) chromophore located in a conserved photosensory core. From recent crystal structures of this core region, it has been inferred that the chromophore structures of P(r) and P(fr) are conserved in most phytochromes. Using circular dichroism spectroscopy and ab initio calculations, we establish that the P(fr) states of the biliverdin-containing bacteriophytochromes DrBphP and PaBphP are structurally dissimilar from those of the phytobilin-containing cyanobacterial phytochrome Cph1. This conclusion is further supported by chromophore substitution experiments using semisynthetic bilin monoamides, which indicate that the propionate side chains perform different functional roles in the 2 classes of phytochromes. We propose that different directions of bilin D-ring rotation account for these distinct classes of red/far-red photochemistry.

Fig. 1.

The P_fr states of Cph1 and DrBphP are distinct. (A) CD spectra of 21 μM Cph1 in the P_r state (solid line) and at P_r/P_fr photoequilibrium (dashed line). (B) CD spectra of 17 μM DrBphP in the P_r state (solid line) and at P_r/P_fr photoequilibrium (dashed line). The far-red absorbance of P_fr is indicated.

Fig. 2.

Apophytochromes incorporate chromophore 12-monoamides. (A) PCB12MA (solid line) was assembled with apoCph1 to give the PCB12MA adduct Cph1-PCB12MA, which has a dual-P_r spectrum (dashed line; peaks indicated). (B) BV12MA (solid line) was assembled with apoDrBphP to give the BV12MA adduct DrBphP-BV12MA, which has a similar dual-P_r spectrum (dashed line; peaks indicated). Spectra are normalized to the blue/UV (Soret) band.

Fig. 3.

Distinct roles for the 12-propionate in Cph1 and DrBphP photochemistry. (A) Cph1-PCB12MA was illuminated with 650 ± 20 nm light (indicated), and spectra were taken as a function of time. (B) The photochemical difference spectrum is plotted for the data in A. (C) DrBphP-BV12MA was illuminated with 670 ± 20 nm light (indicated) as in A. Arrows indicate peak/trough regions in the difference spectrum. (D) Dark reversion of the dual-P_fr photoequilibrium was examined. Arrows are as in C. (E) Difference spectra are shown for conversion of DrBphP-BV12MA from P_r to P_fr with 670 ± 20 nm light (blue), for subsequent conversion of the same sample with 600 ± 5 nm light (green), for conversion of the 670-nm photoequilibrium mixture with 750 ± 20 nm (red), and for dark reversion of the 600-nm photoequilibrium mixture to the P_fr state (purple). (F) Normalized absorbance at 750 nm is shown for conversion of the dual-P_r/dual-P_fr photoequilibrium mixture of DrBphP-BV12MA to dual-P_r either via dark reversion (blue) or in the presence of 750 ± 20 nm light (red). Illum, illumination.

Fig. 4.

Different directions of D-ring rotation in phytochrome photochemistry. (Upper) The ring skeleton for the P_r chromophore is shown. Rotation of the D-ring to a formally E,anti configuration will lead to a strained 15/16 bond. (Lower Left) Counterclockwise rotation of the α-facial D-ring about the 15/16 bond (blue) will result in a steric clash between the indicated methyl groups at C13¹ and C17¹ as the 15/16 dihedral relaxes. This steric repulsion causes the D-ring to slump to the bilin β-face and results in a D-β_f C15-E,anti configuration (Movie S1). (Lower Right) Clockwise rotation of the D-ring (red) does not produce this clash upon 15/16 dihedral relaxation and permits further rotation to a D-α_f C15-E,anti configuration (Movie S2).