PecE and PecF jointly catalyze the covalent attachment of phycocyanobilin to Cys-R84 of PecA and its concomitant isomerization to phycoviolobilin. (a) An Eschertchia coli supernatant expressing pecF has a residual activity of 6%; compared to the holoenzyme, this activity is lost upon purification. (b) Functional domains of both subunits from the cyanobacterium Mastigocladus laminosus were evaluated by mutageneses and chemical modification of amino acids. When in PecE the two motifs Y29YAAWWL and D263DLL were deleted, the holoenzyme lost its activity; it is also inactivated upon deletion of a central part (R111 to A122). The three conserved cysteines C48, C91, and C161 have only minor effects on catalysis. When in PecF the 20 C-terminal and 56 N-terminal amino acids were truncated, the lyaseisomerase activity in combination with PecE decreased to 12% and 15%, respectively, compared to the native enzyme. The catalytic efficiency (kcat/Km) decreased 16-fold when the unique four histidine residues in PecF beginning at H53 were deleted. H121 and C122 of PecF are essential for the enzyme activity; they are part of a unique stretch extending from A104 to N125 which is absent in the β-subunit of related but nonisomerizing lyases. A single histidine and a single tryptophan are equired for activity in both PecE and PecF, as judged from diethyl pyrocarbonate and N-bromosuccinimide modification and statistical analyses. Inactivation of PecE and PecF is also possible by arginine-specific reagents, while modifications of lysine, glutamate, and aspartate retained activity.(c) PecE and PecF, as well as most of the mutants, bind PCB covalently in substoichiometric amounts, as assayed by Zn2+_induced fluorescence on denaturing gels.

ent spectra (maxima of absorption, circular and linear dichroism) of individual chromophores have been assigned for phycoerythrocyanin (PEC) trimer, monomer(s), and its subunits (α-PEC and β-PEC) by titration with p-chloromercury-benzene-sulfonate (PCMS), linear dichroism and photochemical transformations, as well as by deconvolution using a‘bilin’ line-shape spectrum based on the α-84 phycoviolobilin-chromophore in the α-subunit. The level ordering PVB-α-84→PCB-α-155→PCB-α-84 is the same irrespective of aggregation. Two different monomers (αβ) were observed. In 4M urea, the spectra are appropriately weighted sums of the subunit spectra, whereas in the monomer obtained in 1M KSCN, both β-chromophores are red-shifted by 4-5 nm. Formation of trimer (αβ)3 gives considerable spectral changes: (1) the absorption is narrowed, which has been rationalized by excitonic coupling between neighbouring monomers, (2) the short wavelength part in the CD spectrum is missing and (3) a fourth band (+) at 528 in the LD spectrum appears. A deconvolution of the trimeric aggregation state using only the ‘bilin’ line-shape model is not possible.

The core-membrane linker, LCM, connects functionally the extramembraneous light-harvesting complex of cyanobacteria, the phycobilisome, to the chlorophyll-containing core-complexes in the photosynthetic membrane. Genes coding for the apoprotein, ApcE, from Nostoc sp. PCC 7120 and for a C-terminally truncated fragment ApcE(1-240) containing the chromophore binding cysteine-195 were overexpressed in Escherichia coli. Both bind covalently phycocyanobilin (PCB) in an autocatalytic reaction, in the presence of 4M urea necessary to solubilize the proteins. If judged from the intense, red-shifted absorption and fluorescence, both products have the features of the native core-membrane linker LCM, demonstrating that the lyase function, the dimerization motif, and the capacity to extremely red-shift the chromophore are all contained in the N-terminal phycobilin domain of ApcE. The red-shift is, however, not the result of excitonic interactions: Although the chromoprotein dimerizes, the circular dichroism shows no indication of excitonic coupling. The lack of homologies with the autocatalytically chromophorylating phytochromes, as well as with the heterodimeric cysteine-a84 lyases, indicates that ApcE constitutes a third type of bilin: biliprotein lyase.

Photochromic biliproteins can be switched by light between two states, initiated by Z/E photoisomerization of the linear tetrapyrrole chromophore. The cyanobacterium Anabaena sp. PCC 7120 contains three genes coding for such biliproteins, two coding for phytochromes (aphA/B) and one for the R subunit of phycoerythrocyanin (pecA).(a) aphA was overexpressed in Escherichia coli with N-terminal His and S tags, and the protein was reconstituted by an optimized protocol with phycocyanobilin (PCB), to yield the photochromic chromoprotein, PCB-AphA, carrying the PCB chromophore. (b) AphA chromophorylation is autocatalytic such as in other phytochromes. (c) AphA chromophorylation is also possible by chromophore transfer from the PCB-carrying biliprotein, phycocyanin (CPC). The autocatalytic transfer is very slow, and it is enhanced more than 100-fold by catalysis of PCB: CpcA lyase and R-CPC as donor.(d) Through deletion mutations of aphA, a short sequence IQPHGV [amino acids (aa) 26-31] was found essential for the lyase activity of AphA, indicating an interaction of the N terminus with the chromophore-binding domain around cysteine 259. (e) A motif of at least 23 aa, starting with this sequence and located 250 aa N terminal of the chromophore-binding cysteine, is proposed to relate to the lyase function in plant and most prokaryotic phytochromes. (f) Long-range interactions in AphA are further supported by blue-shifted absorptions (e12 nm) of both the Pr and Pfr forms of truncated chromoproteins.

While chromophore attachment to a-subunits of cyanobacterial biliproteins has been studied in some detail, little is known about this process in h-subunits. The ones of phycoerythrocyanin and C-phycocyanin each carry two phycocyanobilin (PCB) chromophores covalently attached to cysteinsh 84 and h155. The differential nonenzymatic reconstitution of PCB to the apoproteins, PecA, PecB, CpcA and CpcB, as well as to mutant proteins of the h-subunits lacking either one of the two binding cysteins, was studied using overexpression of the respective genes. PCB adds selectively to Cys-84 of CpcA, CpcB, PecA, and PecB, but the bound chromophore has a nonnative configuration, and in the case of CpcA, is partly oxidized to mesobiliverdin (MBV). The oxidation is independent of thiols but can be suppressed by ascorbate. The addition to Cys-h84 is suppressed in the presence of detergents like Triton X-100, in favor of an addition to Cys-h155 yielding the correctly bound chromophore. Triton X-100 also inhibits oxidation of the chromophore during addition to CpcA. The effect of Triton X-100 was studied on the isolated components of the reconstitution system. Absorption, fluorescence and circular dichroism spectra indicate a major conformational change of the chromophore upon addition of the detergent, which probably controls the site selectivity of the addition reaction, and inhibits the oxidation of PCB to MBV.

Truncated chromopeptides have been prepared from the small photo-and redox-switchable biliprotein α-phycoerythrocyanin (α-PEC). The native chromoprotein consists of a C-terminal globin domain containing the chromophore and the regulatory cysteins 98 and 99, and a two-helix (X, Y) N-terminal domain responsible for aggregation. Digestion with chymotrypsin-free trypsin leads to three chromopeptides, (N-30, N-33 and N-35), basically lacking the two N-terminal helices X and Y. The photo- and redox chemistry of the major product (N-33) is identical, qualitatively and uantitatively, to that of native α-PEC. A series of N-and C-terminally truncated polypeptides were expressed in E. coli and subjected to autocatalytic and enzymatic reconstitution with phycocyanobilin. Enzymatic reconstitution was possible with N-terminally truncated polypeptides up to 45 aa, while neither a more extensively shortened (N-63) peptide, nor two C-terminally shortened polypeptides could be reconstituted. All chromopeptides recovered from enzymatic reconstitution contained the native phycoviolobilin chromophore and showed the photochemical and redox reactivity of α-PEC, albeit quantitatively reduced in the N-45 chromopeptide.

Cofactor requirements and enzyme kinetics have been studied of the novel, dual-action enzyme, the isomerizing phycoviolobilin phycoerythrocyanin-α84-cystein-lyase (PVBPEC-lyase) from Mastigocladus laminosus, which catalyses both the covalent attachment of phycocyanobilin to PecA, the apo-α-subunit of phycoerythrocyanin, and its isomerization to phycoviolobilin. Thiols and the divalent metals, Mg2+ or Mn2+, were required, and the reaction was aided by the detergent, Triton X-100. Phosphate bu er inhibits precipitation of the proteins present in the reconstitution mixture, but at the same time binds the required metal. Kinetic constants were obtained for both substrates, the chromophore (Km= 12-16μM, depending on [PecA], kcat≈1.2×10-4.s-1) and the apoprotein (Km=2.4μM at 14μM PCB, kcat≈0.8×10-4.s-1). The kinetic analysis indicated that the reconstitution reaction proceeds by a sequential mechanism. By a combination of untagged and His-tagged subunits, evidence was obtained for a complex formation between PecE and PecF (subunits of PVB-PEClyase), and by experiments with single subunits for the prevalent function of PecE in binding and PecF in isomerizing the chromophore.

PecE and PecF, the products of two phycoerythrocyanin lyase genes (pecE and pecF) of Mastigocladus laminosus (Fischerella), catalyze two reactions: (1) the regiospecific addition of phycocyanobilin (PCB) to Cys-R84 of the phycoerythrocyanin R-subunit (PecA), and (2) the △4f△2 isomerization of the PCB to the phycoviolobilin (PVB)-chromophore [Zhao et al. (2000) FEBS Lett. 469, 9-13]. The R-apoprotein (PecA) as well PecE and PecF were overexpressed from two strains of M. laminosus, with and without His-tags. The products of the spontaneous addition of PCB to PecA, and that of the reaction catalyzed by PecE/F, were characterized by their photochemistry and by absorption, fluorescence, circular dichroism of the four states obtained by irradiation with light (15-Z/E isomers of the chromophore) and/or modification of Cys-R98/99 with thiol-directed reagents. The spontaneous addition leads to a 31-Cys-PCB adduct, which is characteristic of allophycocyanins and phycocyanins, while the addition catalyzed by PecE and PecF leads to a 31-Cys-PVB adduct which after purification was identical to R-PEC. The specificity and kinetics of the chromophore additions were investigated with respect to the structure of the bilin substrate: The 3-ethylidene-bilins, viz., PCB, its 18-vinyl analogue phytochromobilin, phycoerythrobilin and its dimethylester, react spontaneously to yield the conventional addition products (3-H, 31-Cys), while the 3-vinyl-substituted bilins, viz., bilirubin and biliverdin, were inactive. Only phycocyanobilin and phytochromobilin are substrates to the addition-isomerization reaction catalyzed by PecE/F. The slow spontaneous addition of phycoerythrobilin is not influenced, and there is in particular no catalyzed isomerization to urobilin.

The structure of phycoviolobilin, the photoactive chromophore of a-phycoerythrocyanin, is incompatible with a chromophore ligation to the apoprotein via SH-addition (cysteine) to a △3, 3 1-double bond of the phycobilin. The two putative phycoerythrocyanin lyase genes of Mastigocladus laminosus, pecE and pecF, were overexpressed in Escherichia coli. Their action has been studied on the addition reaction of phycocyanobilin to apo-K-phycoerythrocyanin (PecA). In the absence of the components of K-PEC-phycoviolobilin lyase PecE and PecF, or in the presence of only one of them, phycocyanobilin binds covalently to PecA forming a fluorescent chromoprotein with a red-shifted absorption (λmax= 641nm) and low photoactivity (＜10%). In the presence of both PecE and PecF, a chromoprotein forms which by its absorption (λmax=565nm) and high photoreversible photochromism (100% type I) has been identified as integral K-phycoerythrocyanin. We conclude that PecE and PecF jointly catalyze not only the addition of phycocyanobilin to PecA, but also its isomerization to the native phycoviolobilin chromophore.