Biliverdin-copper complex at the physiological pH

Abstract

Biliverdin (BV) is a degradation product of heme catabolism, which is rapidly converted to bilirubin (BR) by BV reductase 1. Biliverdin and unconjugated BR, commonly named bile pigments, have important function in biochemical processes. The presence of copper and other biological and toxic transitional metals at significant concentrations in bile implies the possibility that metal complexes with bile pigments can be formed 2. Consequently, our interest was to study the complex of BV with copper in physiological conditions – phosphate buffer with pH 7.4. UV-Vis spectrophotometry was applied to investigate formation/degradation of complex of BV with copper ions and to check stoichiometry by titration, showing that BV interacted with Cu2+ in 1:1 stoichiometry. Mass spectroscopy analysis confirmed this – ion at m/z 643.36 was detected. The results of Raman spectroscopy of BV were in good agreement with previous reports 3. Comparing spectra of BV and BV-Cu complex, the following differences were observed: a new band at low wave number is emerged for the complex may be attributed to Cu-N bond vibration; the band which was shifted to lower energies implicates increased stability of BV in the complex; intensity changes imply a more planar structure of BV in the complex, while stronger bands in complex imply higher delocalization of π-electrons and consequently a higher stability of the BV structure. Pertinent to this, it has been proposed that complexes of BV model compounds with Cu2+ may show unusual electronic structures that exhibit a significant ligand radical character. 1H NMR spectrum of BV in phosphate buffer had a poor resolution of signals, which may originate from aggregation, but this was of little relevance here, since the addition of copper ions led to very strong effect - the complete loss of almost all lines. The loss of signals represents the result of strong paramagnetic effects that may come from an unpaired e- that is delocalized in pπ orbitalss of the ring/ligand influencing all protons in the complex. The EPR spectrum of Cu2+ (S = 1/2; I = 3/2) in phosphate buffer shows that Cu2+ is weakly coordinated in an axial symmetry with one gr line and four lines coming from hyperfine coupling along gs. The addition of BV in equimolar concentration led to the loss of Cu2+ signal. The remaining signal in the [BV]/[Cu2+] = 1 system was broad, and did not show hyperfine structure. The g-value of the isotropic signal of BV-Cu complex was significantly lower than the average g-value of Cu2+ in the phosphate buffer indicating delocalization of the spin away from the metal nucleus. Similar EPR signals have been reported previously 4. Parallel-mode EPR showed no signal. Furthermore, the spectra were run over a wide field range and no half field lines were observed, either in parallel or in perpendicular mode. These results are consistent with S = 0 for the copper center. Further, redox properties of the complex were examined. BV showed a well-defined anodic peak. The [BV]/[Cu2+] = 2 system showed two additional oxidation peaks at much lower potentials than BV. The former potential corresponds to the oxidation of Cu1+, as we have shown previously 5. There was a slight consumption of O2 in [BV]/[Cu2+] = 1 system, which may be explained by traces of ‘free’ copper. However, in the presence of an excess of copper ([BV]/[Cu2+] = 0.5), the consumption of O2 was significant. This implies that ‘free’ Cu2+ reacts with the complex and ‘shuttles’ an e- to O2. The complex was susceptible to oxidizing agents but not to reducing agents. Considering the results obtained we conclude that, at physiological pH, BV builds a complex with copper ions in 1:1 stoichiometry. The formation of complex involves the rearrangement of electronic structure which provides increased energetic stability and strong paramagnetic effects. We believe that a complex with a highly delocalized unpaired e- and the formal BV•+-Cu1+ character best suites the outlined properties, but other structures of the complex cannot be completely ruled out. The presented results may shed new light on long-standing issues of BV chemistry and catalysis in biological systems.