Biliverdin Binding Research Articles

The effect of pH on the initial rate kinetics of the dimeric biliverdin‐IXα reductase from the cyanobacterium Synechocystis PCC6803

Biliverdin-IXalpha reductase from Synechocystis PCC6803 (sBVR-A) is a stable dimer and this behaviour is observed under a range of conditions. This is in contrast to all other forms of BVR-A, which have been reported to behave as monomers, and places sBVR-A in the dihydrodiol dehydrogenase/N-terminally truncated glucose-fructose oxidoreductase structural family of dimers. The cyanobacterial enzyme obeys an ordered steady-state kinetic mechanism at pH 5, with NADPH being the first to bind and NADP(+) the last to dissociate. An analysis of the effect of pH on k(cat) with NADPH as cofactor reveals a pK of 5.4 that must be protonated for effective catalysis. Analysis of the effect of pH on k(cat)/K(m)(NADPH) identifies pK values of 5.1 and 6.1 in the free enzyme. Similar pK values are identified for biliverdin binding to the enzyme-NADPH complex. The lower pK values in the free enzyme (pK 5.1) and enzyme-NADPH complex (pK 4.9) are not evident when NADH is the cofactor, suggesting that this ionizable group may interact with the 2'-phosphate of NADPH. His84 is implicated as a crucial residue for sBVR-A activity because the H84A mutant has less than 1% of the activity of the wild-type and exhibits small but significant changes in the protein CD spectrum. Binding of biliverdin to sBVR-A is conveniently monitored by following the induced CD spectrum for biliverdin. Binding of biliverdin to wild-type sBVR-A induces a P-type spectrum. The H84A mutant shows evidence for weak binding of biliverdin and appears to bind a variant of the P-configuration. Intriguingly, the Y102A mutant, which is catalytically active, binds biliverdin in the M-configuration.

The use of synthetic linear tetrapyrroles to probe the verdin sites of human biliverdin‐IXα reductase and human biliverdin‐IXβ reductase

Many vertebrate species express two enzymes that are capable of catalysing the reduction of various isomers of biliverdin. Biliverdin-IXalpha reductase (BVR-A) is most active with its physiological substrate biliverdin-IXalpha, but can also reduce the three other biliverdin isomers IXbeta, IXdelta and IXgamma. Biliverdin-IXbeta reductase (BVR-B) catalyses the reduction of only the IXbeta, IXdelta and IXgamma isomers of biliverdin. Therefore, the activity of BVR-A can be measured using biliverdin-IXalpha as a specific substrate. We now show that the dimethyl esters of biliverdin-IXbeta and biliverdin-IXdelta are substrates for BVR-B, but not for BVR-A. This provides a useful method for specifically assaying the activity of both BVR-A and BVR-B in crude mixtures, using biliverdin-IXalpha for BVR-A and the dimethyl ester of either biliverdin-IXbeta or biliverdin-IXdelta for BVR-B. Human BVR-A has been suggested as a pharmacological target for neonatal jaundice. Because of the absence of a crystal structure with biliverdin bound to BVR-A, we have investigated indirect ways of examining tetrapyrrole binding. In the present study, we report that a number of sterically locked conformers of 18-ethylbiliverdin-IXalpha are substrates for human BVR-A, and discuss the implications for the biliverdin binding site. The oxidation of bilirubin-IXalpha ditaurate to biliverdin-IXalpha ditaurate is also described. We show that biliverdin-IXalpha ditaurate is a substrate for human BVR-A and discuss the possibility of using a competing substrate, which is reduced to a water soluble and excretable rubin, as a prototypic inhibitor of BVR-A.

Characterization of the Covalent and Noncovalent Adducts of Agp1 Phytochrome Assembled with Biliverdin and Phycocyanobilin by Circular Dichroism and Flash Photolysis

The functional role of the covalent attachment of the bilin chromophores biliverdin (BV) and phycocyanobilin (PCB) was investigated for phytochrome Agp1 from Agrobacterium tumefaciens using circular dichroism (CD) and transient absorption spectroscopy. Covalent and noncovalent adducts with these chromophores were prepared by using wild-type (WT) Agp1 (covalent BV and noncovalent PCB binding), mutant C20A in which the covalent BV binding site is eliminated, and mutant V249C in which the covalent PCB binding site is introduced. While the CD spectra of the P(r) forms of all these photochromic adducts are qualitatively the same, the CD spectrum of the P(fr) form of the covalent PCB adduct is unique in having a positive rotational strength in the Q-band which we assign to the Z-E isomerization of the C-D methine bridge. In the three other adducts, the Q-band CD in the P(fr) state is almost zero, suggesting that upon photoconversion a negative contribution from an out-of-plane rotation of the A ring of the chromophore compensates for the positive contribution from ring D. The contribution from ring A is absent or strongly reduced in the shorter pi-conjugation system of the covalent PCB adduct. The results from CD spectroscopy are consistent with a uniform geometry of the bilin chromophore in the covalent and noncovalent adducts. Transient absorption spectroscopy showed that the spectral changes and the kinetics of the P(r) to P(fr) photoconversion are not substantially affected by the covalent attachment of BV and PCB. The kinetics in the BV and PCB adducts mainly differ in the formation of P(fr) that is accelerated by 2 orders of magnitude in the PCB adducts, whereas the sequence of spectral transitions and the associated proton transfer processes are quite similar. We conclude that the P(r) to P(fr) photoconversion in the BV and PCB adducts of Agp1 involves the same relaxation processes and is thus governed by specific protein-cofactor interactions rather than by the chemical structure of the chromophore or the mode of attachment. The strongly reduced photostability of the noncovalent BV adduct suggests that covalent attachment in native Agp1 phytochrome prevents irreversible photobleaching and stabilizes the chromophore. The N-terminal peptide segment including amino acids 2-19 is essential for covalent attachment of the chromophore but dispensable for the spectral and kinetic properties of Agp1.

Biliverdin is the endogenous ligand of human serum α 1-acid glycoprotein

α 1-Acid glycoprotein (AAG), an acute phase component of the human serum, is a prominent member of the lipocalin family of proteins showing inflammatory/immunomodulatory activities and promiscuous drug binding properties. Both three-dimensional structure of AAG and its precise biological function are still unknown and only a few endogenous AAG ligands have been described to date. CD spectroscopic studies performed with commercial AAG and the separated genetic variants revealed high-affinity binding of biliverdin (BV) and biliverdin dimethyl ester to the ‘F1/S’ fraction of the protein. The preferential accommodation of the right-handed, P-helicity conformers of the pigments by the protein matrix resulted in strong induced CD activity, which was utilized for estimation of the binding parameters and to locate the binding site. It was concluded that both pigments are bound in the central β-barrel cavity of AAG, held principally by hydrophobic interactions. Possible biological implications of the BV binding ability of AAG with special emphasis on the heme oxygenase-1 pathway are discussed.

Sub‐Picosecond Mid‐Infrared Spectroscopy of Phytochrome Agp1 from Agrobacterium tumefaciens

The photoinduced primary reaction of the biliverdin binding phytochrome Agp1 (Agp1-BV) from Agrobacterium tumefaciens was investigated by sub-picosecond time-resolved Vis pump-IR probe spectroscopy. Three time constants of tau(1)=0.7+/-0.05 ps, tau(2)=3.3+/-0.2 ps and tau(3)=33.3+/-1.5 ps could be isolated from the dynamics of structurally specific marker bands of the BV chromophore. These results together with those of accompanying sub-picosecond Vis pump-Vis probe spectroscopy allow the extension of the reaction scheme for the primary process by a vibrationally excited electronic ground state. The isomerization at the C15=C16 bond occurs within the lifetime of the excited electronic state. A quantum yield of 0.094 for the primary reaction is determined, suggesting that the quantum yield of formation of the P(fr) far-red-absorbing form is already established in the primary photoreaction of the P(r) (red-absorbing) form.

Evolution of a bacteriophytochrome from light to redox sensor

Bacteriophytochromes are red/far-red photoreceptors that bacteria use to mediate sensory responses to their light environment. Here, we show that the photosynthetic bacterium Rhodopseudomonas palustris has two distinct types of bacteriophytochrome-related protein (RpBphP4) depending upon the strain considered. The first type binds the chromophore biliverdin and acts as a light-sensitive kinase, thus behaving as a bona fide bacteriophytochrome. However, in most strains, RpBphP4 does not to bind this chromophore. This loss of light sensing is replaced by a redox-sensing ability coupled to kinase activity. Phylogenetic analysis is consistent with an evolutionary scenario, where a bacteriophytochrome ancestor has adapted from light to redox sensing. Both types of RpBphP4 regulate the synthesis of light harvesting (LH2) complexes according to the light or redox conditions, respectively. They modulate the affinity of a transcription factor binding to the promoter regions of LH2 complex genes by controlling its phosphorylation status. This is the first complete description of a bacteriophytochrome signal transduction pathway involving a two-component system.

Investigation of the association behaviors between biliverdin and bovine serum albumin by fluorescence spectroscopy

The interaction between biliverdin and bovine serum albumin (BSA) has been studied by steady fluorescence spectroscopy, synchronous fluorescence and resonance light scanning spectra. The binding of biliverdin to BSA quenches the tryptophan residue fluorescence and the results show that both static and dynamic quenching occur together with complex formation. The binding constant and binding sites of biliverdin to BSA at pH 7.1 are calculated to be 3.33 × 10 8 L/mol and 1.54, respectively, according to the double logarithm regression curve. In addition, the distance between the biliverdin and BSA is estimated to be 1.25 nm using Föster's equation on the basis of the fluorescence energy transfer. Furthermore the synchronous fluorescence spectra show that the microenvironment of the tryptophan residues has not obvious changes, which obeys the phase distribution model. Finally, the thermodynamic data show that biliverdin molecules enter the hydrophobic cavity of BSA via hydrophobic interaction.

Sterically Locked Synthetic Bilin Derivatives and Phytochrome Agp1 from Agrobacterium tumefaciens Form Photoinsensitive Pr- and Pfr-like Adducts

Phytochrome photoreceptors undergo reversible photoconversion between the red-absorbing form, Pr, and the far-red-absorbing form, Pfr. The first step in the conversion from Pr to Pfr is a Z to E isomerization around the C15=C16 double bond of the bilin chromophore. We prepared four synthetic biliverdin (BV) derivatives in which rings C and D are sterically locked by cyclizing with an additional carbon chain. In these chromophores, which are termed 15Za, 15Zs, 15Ea, and 15Es, the C15=C16 double bond is in either the Z or E configuration and the C14-C15 single bond in either the syn or anti conformation. The chromophores were assembled with Agrobacterium phytochrome Agp1, which incorporates BV as natural chromophore. All locked BV derivatives bound covalently to the protein and formed adducts with characteristic spectral properties. The 15Za adduct was spectrally similar to the Pr form and the 15Ea adduct similar to the Pfr form of the BV adduct. Thus, the chromophore of Agp1 adopts a C15=C16 Z configuration and a C14-C15 anti conformation in the Pr form and a C15=C16 E configuration and a C14-C15 anti conformation in the Pfr form. Both the 15Zs and the 15Es adducts absorbed only in the blue region of the visible spectra. All chromophore adducts were analyzed by size exclusion chromatography and histidine kinase activity to probe for protein conformation. In either case, the 15Za adduct behaved like the Pr and the 15Ea adduct like the Pfr form of Agp1. Replacing the natural chromophore by a locked 15Ea derivative can thus bring phytochrome holoprotein in the Pfr form in darkness. In this way, physiological action of Pfr can be studied in vivo and separated from Pr/Pfr cycling and other light effects.

The pair of bacteriophytochromes from Agrobacterium tumefaciens are histidine kinases with opposing photobiological properties

Bacteriophytochrome photoreceptors (BphPs) are a family of phytochrome-like sensor kinases that help a wide variety of bacteria respond to their light environment. In Agrobacterium tumefaciens, a unique pair of BphPs with potentially opposing roles in light sensing are present. Both AtBphPs contain an N-terminal chromophore-binding domain that covalently attaches a biliverdin chromophore. Whereas AtBphP1 assumes a Pr ground state, AtBphP2 is unusual in that it assumes a Pfr ground state that is produced nonphotochemically after biliverdin binding through a transient Pr-like intermediate. Photoconversion of AtBphP2 with far-red light then generates Pr but this Pr is also unstable and rapidly reverts nonphotochemically to Pfr. AtBphP1 contains a typical two-component histidine kinase domain at its C terminus whose activity is repressed after photoconversion to Pfr. AtBphP2 also functions as a histidine kinase but instead uses a distinct two-component kinase motif that is repressed after photoconversion to Pr. We identified sequences related to this domain in numerous predicted sensing proteins in A. tumefaciens and other bacteria, indicating that AtBphP2 might represent the founding member of a family of histidine phosphorelay proteins that is widely used in environmental signaling. By using these mutually opposing BphPs, A. tumefaciens presumably has the capacity to simultaneously sense red light-rich and far-red light-rich environments through deactivation of their associated kinase cascades.

Purification and characterization of biliverdin-associated cyanoprotein from eggs and hemolymph of the bean bug, Riptortus clavatus (Heteroptera: Alydidae)

Biliverdin binding protein, cyanoprotein (CP), was purified from egg extracts of the bean bug, Riptortus clavatus. The preparation was shown to be homogeneous by DEAE-chromatography and polyacrylamide gel electrophoresis (PAGE). The native molecular weight (MW) of egg CP (CP egg) was estimated to be 320,000 by PAGE and 335,000 by gel filtration. The apoprotein consisted of identical subunits with MW of 76,000. CP egg had a high content of aromatic amino acids (17% mol ratio) with absorbance peaks at 370 and 620 nm which are characteristic of biliverdins. Native CP egg was associated non-covalently with 16 molecules of biliverdin. CP egg contained about 13.0% neutral sugar (mainly mannose) associated covalently with the apoprotein, no lipid was detected. In the hemolymph of the bugs, four CP bands (CP1, 2, 3 and 4) were detected by native PAGE. These bands were immunologically related and showed properties similar to CP egg in biochemical analysis. Only CP1 was identical to CP egg. CPs specific to the hemolymph, CP2, 3 and 4, were different from CP egg in mobility by PAGE, pI and peptide mapping profiles.

The reaction mechanism of bovine kidney biliverdin reductase

The steady-state kinetics of biliverdin reductase can be studied in detail at pH 9 as under these conditions the K m for biliverdin is high enough to obtain reliable measurements of the initial rate in the absence of any biliverdin binding proteins. The initial rate kinetics and the product-inhibition studies are consistent with an ordered sequential mechanism provided the biliverdin concentration was below 20 μM. Above this concentration significant flux occurs through a substrate inhibition pathway involving an enzyme-NAD(P)-biliverdin complex. Chloride is shown to cause a significant activation of the enzyme under certain conditions and this is shown to result from an inability of biliverdin to bind to an enzyme-NAD-chloride complex.

Kinetic properties and regulation of biliverdin reductase

In kinetic studies of the dual nucleotide enzyme biliverdin reductase, product inhibition patterns obtained with bilirubin as the inhibitor, using either the NADPH-linked reaction at pH 8.7 or the NADH-linked reaction at pH 7.0, are consistent with a random order of substrate addition and product release at either pH. With NAD(P) as the product inhibitor, the inhibition patterns suggest the existence of an enzyme-NAD(P)-biliverdin abortive complex. In the absence of added products, substrate inhibition observed at high biliverdin concentrations is also consistent with such a complex. Direct evidence for the existence of enzyme-NADP-biliverdin abortive complexes has been obtained at both pH 7.0 and 8.7 using the fluorescent NADP analog nicotinamide-1- N-6-ethenoadenine dinucleotide phosphate. Etheno-NADP fluorescence is enhanced in enzyme:etheno-NADP complexes but quenched in enzyme:biliverdin:etheno-NADP complexes. The effects of iron hematoporphyrin and cobalt protoporphyrin on the activity of biliverdin reductase have been examined. Iron-hematoporphyrin is a competitive inhibitor with respect to coenzyme at either pH. Comparison of K m values for biliverdin and K i values for bilirubin or iron-hematoporphyrin indicate that while the binding of biliverdin is relatively pH independent, bilirubin binds more tightly at pH 7.0 than pH 8.7. Conversely, Fe-hematoporphyrin binds considerably tighter at pH 8.7 than pH 7.0. With cobalt-protoporphyrin, however, activation is observed, suggesting the existence of a regulatory site on biliverdin reductase for porphyrin derivatives. Resonance energy transfer measurements, from the fluorescent coenzyme analog etheno-NADP to biliverdin, iron-hematoporphyrin, or cobalt-protoporphyrin indicate that while the biliverdin site is located approximately 15 Å from the etheno moiety of the coenzyme, both porphyrin derivatives are located 23.5 Å away. This suggests the existence of a separate porphyrin binding site on biliverdin reductase which, depending upon the nature of the porphyrin binding, can produce activation or inhibition of the enzyme.

Purification and characterization of a biliverdin-associated protein from the hemolymph of Manduca sexta.

A biliverdin binding protein, insecticyanin, has been isolated from the hemolymph of the fourth instar tobacco hornworm Manduca sexta. The protein has been purified to apparent homogeneity by conventional chromatography with a cumulative yield of 40-50%. The protein (Mw 71 600) is composed of three subunits (Mr 23 000). Each subunit binds one biliverdin molecule. Proton magnetic resonance spectroscopy and absorption spectroscopy demonstrate that the bilin is the biliverdin IX gamma isomer.

An isotachophoretic analysis of the interaction of bilirubin and biliverdin with bovine serum albumin

AbstractThe binding sites of non‐defatted and fatty acid‐free bovine serum albumin for bilirubin and biliverdin were investigated by means of analytical capillary isotachophoresis. In addition, the effect of oleic acid on the binding of bilirubin was examined with the same analytical tool. Albumin‐bilirubin complexes with increasing molar ratios of bilirubin can be identified as a separately migrating zone adjacent to the zone of free bilirubin. At pH 9 there are 3–4 high‐affinity binding sites for bilirubin and, possibly, several weaker binding sites. Fatty acid‐free and non‐defatted albumin exhibit almost identical binding properties with regard to bilirubin. Under the same conditions non‐defatted albumin shows only one high‐affinity binding site for biliverdin. The analysis of fatty acid‐free albumin‐biliverdin complexes reveals additional weak binding sites, which indicates that the physiological loading of albumin with ligands interferes only with the low‐affinity binding of biliverdin. At sufficiently high concentrations, oleic acid even competes for the first high‐affinity binding site of bovine serum albumin for bilirubin. There is a linear relationship between the zone length and the molar concentrations of free ligands (bilirubin, biliverdin). The differential binding characteristics of bilirubin and biliverdin indicate that the reduction of biliverdin yields a molecule which has a higher affinity for albumin; this, however, results in an inferior water solubility at physiological pH values. Owing to high resolution and quantification, capillary isotachophoresis is a powerful tool for screening endogenous and exogenous compounds with regard to their effect on the bilirubin transport capacity of albumin.

Bilirubin and biliverdin binding to raty protein (ligandin)

The binding of bilirubin to poly( l-lysine) produces an optically active complex at pH 10.1. Circular dichroism spectra of these complexes are distinguishable from those generated by binding of bilirubin to the high affinity sites on albumin. Comparison of the circular dichroism spectra of bilirubin bound to the hepatic protein ligandin with those of bilirubin complexed with albumin or polylysine indicates that binding of bilirubin to ligandin occurs at two types of sites. These are distinguishable on the basis of their spectral properties, one resembling the high affinity site of bovine serum albumin and the other resembling polylysine. Complexes of biliverdin with albumin and ligandin bear similarities to the bilirubin-protein complexes. The native protein itself has an ordered structure which consists of 41% α-helix and is not altered by the binding of bilirubin.