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The unmediated electrochemistry of two large Cu-containing proteins, ascorbate oxidase and laccase, was investigated by direct-current cyclic voltammetry. Rapid heterogeneous electron transfer was achieved in the absence of promoters or mediators by trapping a small amount of protein within a solid, electrochemically inert, tributylmethyl phosphonium chloride membrane coating a gold electrode. The problems typical of proteins in solution, such as adsorption on the electrode surface, were avoided by this procedure. In anaerobic conditions, the cyclic voltammograms, run at a scan rate of up to 200 mV/s, showed the electron transfer process to be quasi-reversible and diffusion-controlled. The pH-dependent redox potentials (+360 mV and +400 mV against a normal hydrogen electrode at pH7.0 for ascorbate oxidase and laccase respectively and +390 mV and +410 mV at pH5.5) were similar to those of the free proteins. The same electrochemical behaviour was recorded for the type 2 Cu-depleted derivatives, which contain reduced type 3 Cu, whereas the apoproteins were electrochemically inactive. Under aerobic conditions the catalytic current intensity of holoprotein voltammograms increased up to approx. 2-fold at a low scanning rate, with unchanged redox potentials. The voltammograms of type 2 Cu-depleted proteins and of apoproteins were unaffected by the presence of oxygen. This suggests that electron uptake at the electrode surface involves type 1 Cu and that only in the presence of oxygen is the intramolecular electron transfer to other protein sites rapid enough to be observed. The analogy with available kinetic results is discussed.

Aromatic hydrazides of the general formula NH2NHCO(CH2)nC6H4R were covalently bound by bovine serum amine oxidase (BSAO), giving rise to optical and CD absorptions at 350-400 nm. Benzohydrazides (n = 0) reacted slowly, in the ratio of one per dimeric protein molecule, like semicarbazide. Phenylacetohydrazides (n = 1) and phenylpropionic hydrazides (n = 2) reacted instead in the ratio of two per dimer, one molecule at a much faster rate than the other. The fast reaction correlated with the loss of enzymatic activity. The contribution to the optical absorbance of either molecule was identical, but only the first one produced a CD band, the wavelength and sign of which were determined by the number n of methylene groups in the hydrazide. In n = 1 and n = 2 compounds, the reaction was faster as the R substituent became more hydrophobic (triazolyl less than imidazolyl less than phenyl), suggesting a specific interaction with the protein matrix. Phenylhydrazine was found to react with the native enzyme in the ratio of only one per protein dimer. However, one phenylhydrazine was also slowly bound by most 1:1 enzyme-hydrazide adducts, with the formation of ternary derivatives. Phenylhydrazine formed the usual intense band at 447 nm with n = 1 and n = 2 hydrazide-BSAO adducts and a weaker, blue-shifted band with the adducts of semicarbazide and of some n = 0 hydrazides. In both cases, the hydrazide absorption band was unaffected. Competition was observed with other benzohydrazides and with the second molecule of n = 1 compounds. A half-site mechanism appears to be operative, the second site being always less reactive than the first. Reactivity and adduct conformation were also affected by N,N-diethyldithiocarbamate, a powerful enzyme inhibitor that binds copper.

No abstract

The organic cofactor of bovine serum amine oxidase was identified as 2,4,5-trihydroxyphenylalanine quinone by means of the phenylhydrazine adduct [Janes, S. M., Mu, D., Wemmer, D., Smith, A. J., Kaur, S., Maltby, D., Burligame, A.L. & Klinman, J.P. (1990) Science 248, 981-987]. A still debated question is, however, whether the dimeric protein binds two mol phenylhydrazine/mole or only one, that is whether it actually contains two identical independent carbonyl cofactors. This matter is addressed in the present study by means of the protein reactions with phenylhydrazine and other inhibitors such as semicarbazide and p-pyridine-2-yl-phenylacetohydrazide. The two latter reagents were found to bind in two steps, one mole/mole dimer in the first step with loss of catalytic activity but only about (0.10-0.35 mol/mol) in the second one. Similar results were obtained by either optical spectroscopy or by reverse-phase HPLC of the labelled peptides produced on proteolysis. Irrespective of the inhibitor nature and reacted amount, all adducts formed on proteolysis a single labelled peptide, of same 25-amino-acid composition, showing that the same cofactor is present in both subunits, in the same stretch of the polypeptide chain. The slow reaction of the second cofactor may be related to slow conformational equilibria, which are established after the first cofactor has reacted and are probably mediated by a change of the hydrogen bond pattern. The conformers spectroscopic properties suggest that they differ in whether the cofactor does or does not directly interact with copper.