Biliverdin amides reveal roles for propionate side chains in bilin reductase recognition and in holophytochrome assembly and photoconversion

Abstract

Linear tetrapyrroles (bilins) perform important antioxidant and light-harvesting functions in cells from bacteria to humans. To explore the role of the propionate moieties in bilin metabolism, we report the semisynthesis of mono- and diamides of biliverdin IXalpha and those of its non-natural XIIIalpha isomer. Initially, these were examined as substrates of two types of NADPH-dependent biliverdin reductase, BVR and BvdR, and of the representative ferredoxin-dependent bilin reductase, phycocyanobilin:ferredoxin oxidoreductase (PcyA). Our studies indicate that the NADPH-dependent biliverdin reductases are less accommodating to amidation of the propionic acid side chains of biliverdin IXalpha than PcyA, which does not require free carboxylic acid side chains to yield its phytobilin product, phycocyanobilin. Bilin amides were also assembled with BV-type and phytobilin-type apophytochromes, demonstrating a role for the 8-propionate in the formation of the spectroscopically native P(r) dark states of these biliprotein photosensors. Neither ionizable propionate side chain proved to be essential to primary photoisomerization for both classes of phytochromes, but an unsubstituted 12-propionate was required for full photointerconversion of phytobilin-type phytochrome Cph1. Taken together, these studies provide insight into the roles of the ionizable propionate side chains in substrate discrimination by two bilin reductase families while further underscoring the mechanistic differences between the photoconversions of BV-type and phytobilin-type phytochromes.

Figure 1

Synthesis of bilin 8-monoamides. BR13 and BR13 dimethyl ester were co-acid-scrambled to yield mixed rubins including BR-12MME. Amidation of free propionate sidechains and subsequent oxidation yielded mixed verdins including BV-8MA12MME. Ester cleavage and purification yielded BV-8MA, which could be enzymatically converted to PCB-8MA by the ferredoxin-dependent bilin reductase PcyA.

Figure 2

Reaction of BV with PcyA. BV derivatives were analyzed by RP-HPLC in 50% aqueous acetone/20 mM formic acid either with (red) or without (blue) prior treatment with PcyA. Absorbance at 650 nm was monitored using a diode-array detector. Top, reduction of BV gave rise to a mix of 3E-PCB and 3Z-PCB, as expected (52). Second from top, reduction of BV-8MA gave rise to two product peaks (a and b) identified as 3E-PCB-8MA and 3Z-PCB-8MA, respectively. Third from top, reduction of BV-12MA produced products c and d, similarly identified as 3E-PCB-12MA and 3Z-PCB-12MA. Absorbance spectra for 3E- and 3Z-PCB and products a-d are presented in Supp. Fig. 6, and peak wavelengths are reported in Supp. Table 5. Bottom, reduction of BV-DA by PcyA resulted in formation of several apparent products that were not well resolved in this mobile phase. Further characterization of these products (Supp. Fig. 7) suggested the presence of PCB diamide, but it was not possible to obtain pure products.

Figure 3

Assembly of apophytochromes with bilin 8-monoamides. (A) Absorbance spectra are shown for free BV (blue) and BV assembled with DrBphP and then dialyzed to remove unincorporated chromophore (DrBphP:BV, red) Starting amounts of BV were equal. (B) Absorbance spectra are shown for assembly of BV-8MA with DrBphP. Colors and sample handling are as in (A). (C) Adducts formed by assembly of DrBphP with BV (blue), BV-8MA (red), and BV-12MA (green) were analyzed by CD spectroscopy. (D) Absorbance spectra are shown for assembly of Cph1 with PCB. Colors, relative concentrations, and sample handling are as in (A). (E) Absorbance spectra are shown for assembly of Cph1 with PCB-8MA. Colors and sample handling are as in (A). (F) Adducts formed by assembly of Cph1 with PCB (blue), PCB-8MA (red), and PCB-12MA (green) were analyzed by CD spectroscopy.

Figure 4

Photochemistry of DrBphP assembled with bilin monoamides. DrBphP:BV-12MA (A-C) and DrBphP:BV8-MA (D-F) were characterized by absorbance and CD spectroscopy. (A) DrBphP:BV-12MA was converted from dual-P_r (blue) to dual-P_fr (red) with 600 nm light (±5 nm), giving the difference spectrum shown in (B). (C) Both dual-P_r (blue) and dual-P_fr (red) were characterized by CD spectroscopy. Inversion of CD upon photoconversion was not observed, with both dual-P_fr red-band peaks exhibiting negative CD. (D) DrBphP:BV-8MA (blue) was illuminated with 670 nm light (± 20 nm), giving enhanced red absorbance and reduced blue absorbance (red). (E) The difference spectrum is shown for the spectra presented in (D). (F) Both the DrBphP:BV-8MA dark state (blue) and photoproduct (red) were characterized by CD spectroscopy. The photoproduct region corresponding to the P_fr state of DrBphP:BV was associated with negative CD, indicating no inversion of CD occurred. The photoproduct CD peak at shorter wavelength does not match the lineshape of the corresponding absorbance peak well (compare D), and the two peak wavelengths are not in very good agreement (Supp. Table 6). Assignment of this peak is therefore uncertain, as discussed in the text.

Figure 5

Denaturation analysis of DrBphP adducts. (A) DrBphP:BV was denatured in acidic guanidinium chloride in the native P_r (blue) or P_fr (green) states. Illumination of the denatured P_fr state with 600±5 nm light resulted in formation of a product (red) whose absorbance spectrum was equivalent to that of the denatured P_r sample. Similar results could be obtained by illumination of a denatured P_fr sample with 650 ±20 nm light (not shown). (B) Similar results were obtained with DrBphP:BV-12MA. Here, illumination with 650±20 nm light is shown; illumination with 600±5 nm light (not shown) gave similar results. (C) Similar experiments were performed with DrBphP:BV-8MA. The denatured P_fr DrBphP:BV-8MA seemed to be a mix of the denatured P_r and P_fr spectra seen with other adducts, suggesting that primary photochemistry was less efficient with DrBphP:BV-8MA than with other adducts. (D) Difference spectra are shown for photoconversion of the denatured P_fr adducts of DrBphP:BV (blue), DrBphP:BV-12MA (green), and DrBphP:BV-8MA (red).

Figure 6

Photochemistry of Cph1 assembled with PCB monoamides. (A) Cph1:PCB-8MA (blue) was illuminated with 650±20 nm light to form photoproducts (red). (B) The difference spectrum for 650±20 nm illumination (blue) revealed appearance of products at 628 and 705 nm (Supp. Table 6). The difference spectra obtained upon illumination with 600±5 nm light (green) displayed different product ratios. (C) Cph1:PCB-12MA (blue) was illuminated with 650±20 nm light to form photoproducts (red). (D) The difference spectra obtained upon illumination of Cph1:PCB-12MA with 650±20 nm light (blue) or 550±35 nm light (green) indicated the presence of comparable changes.

Figure 7

Denaturation analysis of Cph1 adducts. (A) Cph1:PCB was denatured in acidic guanidinium chloride in the native P_r (blue) or P_fr (green) states. Illumination of the denatured P_fr state with 600±5 nm light resulted in formation of a product (red) whose absorbance spectrum was equivalent to that of the denatured P_r sample. (B) Similar results were obtained with Cph1:PCB-12MA. The apparent absorbance peak at approximately 800 nm in (A) and (B) is associated with a contaminant in some lots of guanidine hydrochloride and is photochemically inert (not shown). (C) Similar results were obtained with Cph1:PCB-8MA. (D) Difference spectra are shown for photoconversion of the denatured P_fr adducts of Cph1:PCB (blue), Cph1:PCB-12MA (green), and Cph1:PCB-8MA (red).