Correlating the structures and activities of the resting oxidized and native intermediate states of a small laccase by paramagnetic NMR.

Multicopper oxidases (MCO) are the fastest and most efficient known catalysts of the oxygen-reduction reaction. When all four copper ions are oxidized during catalysis, the native intermediate state (NI) decays in seconds to the resting oxidized state (RO), which returns to the catalytic cycle via reduction, but at a much slower rate than NI. We report the long-lived (months at 4°C) NI state of the small laccase (SLAC) MCO and the subsequent characterization of both its RO and NI states by paramagnetic (1)H NMR. We find that the RO state of the trinuclear cluster (TNC) is best described as an isolated Type-3 dicopper site, antiferromagnetically coupled by a hydroxo group with -2J=500cm(-1). The NI state is more complicated; we develop a theoretical treatment for the case in which all three copper ions in the TNC are coupled, and find that the results are consistent with three coupling constants of -2J=300, 240, and 160cm(-1). These couplings result in a ground doublet state, a low-lying excited doublet state at 121cm(-1), and a quartet excited state at 411cm(-1), in good agreement with DFT models in which the Type-2 copper has a terminal hydroxo ligand.

Modulation of the electrochemical behavior of tyrosyl radicals by the electrode surface.

The ability to adsorb proteins and enzymes on electrode surfaces enhances opportunities for studying enzyme activity and redox-based catalysis. Proteins may be bound in a chosen orientation on surfaces so that specific sites within them may be preferentially studied, but to date no systematic study of a redox moiety from solvent to electrode surface to the protein milieu has been performed. We report the redox and ionization behavior of tyrosine-cysteine, using the cysteine residue to form covalent linkages with Au and self-assembled-monolayer (SAM)-modified Au surfaces and using the tyrosine for redox activity. In addition, the same redox fragment incorporated into a protein bound to a SAM is examined. We find that directly binding the dipeptide to Au causes the greatest change in properties, while binding it to the SAM causes a slight perturbation in redox potential and a significant perturbation in pK(a). When azurin with a surface-exposed tyrosine is bound to a SAM-modified electrode, the redox potential and pK(a) of the tyrosine are nearly unperturbed from the values found for the dipeptide free in solution. Finally, quantification of the voltammetric signal indicates that tyrosine oxidation in the protein triggers the additional oxidation of another nearby amino acid.

Spectroscopic characterization of a high-potential lipo-cupredoxin found in Streptomyces coelicolor.

For many streptomycetes, a distinct dependence on the "bioavailability" of copper ions for their morphological development has been reported. Analysis of the Streptomyces coelicolor genome reveals a number of gene products encoding for putative copper-binding proteins. One of these appears as an unusual copper-binding protein with a lipoprotein signal sequence and a cupredoxin-like domain harboring a putative Type-1 copper-binding motif. Cloning of this gene from S. coelicolor and subsequent heterologous expression in Escherichia coli has allowed for a thorough spectroscopic interrogation of this putative copper-binding protein. Optical and electron paramagnetic resonance spectroscopies have confirmed the presence of a "classic" Type-1 copper site with the axial ligand to the copper a methionine. Paramagnetic NMR spectroscopy on both the native Cu(II) form and Co(II)-substituted protein has yielded active-site structural information, which on comparison with that of other cupredoxin active sites reveals metal-ligand interactions most similar to the "classic" Type-1 copper site found in the amicyanin family of cupredoxins. Despite this high structural similarity, the Cu(II)/(I) midpoint potential of the S. coelicolor protein is an unprecedented +605 mV vs normal hydrogen electrode at neutral pH (amicyanin approximately +250 mV), with no active-site protonation of the N-terminal His ligand observed. Suggestions for the physiological role/function of this high-potential cupredoxin are discussed.

Characterization of SLAC: a small laccase from Streptomyces coelicolor with unprecedented activity.

Laccases and other four-copper oxidases are usually constructed of three domains: Domains one and three house the copper sites, and the second domain often helps form a substrate-binding cleft. In contrast to this arrangement, the genome of Streptomyces coelicolor was found to encode a small, four-copper oxidase that lacks the second domain. This protein is representative of a new family of enzymes--the two-domain laccases. Disruption of the corresponding gene abrogates laccase activity in the growth media. We have recombinantly expressed this enzyme, called SLAC, in Escherichia coli and characterized it. The enzyme binds four copper ions/monomer, and UV-visible absorption and EPR measurements confirm that the conserved type 1 copper site and trinuclear cluster are intact. We also report the first known paramagnetic NMR spectrum for the trinuclear copper cluster of a protein from the laccase family. The enzyme is highly stable, retaining activity as a dimer in denaturing gels after boiling and SDS treatment. The activity of the enzyme against 2,6-dimethoxyphenol (DMP) peaks at an unprecedentedly high pH (9.4), whereas the activity against ferrocyanide decreases with pH. SLAC binds negatively charged substrates more tightly than positively charged or uncharged molecules.

An outer-sphere hydrogen-bond network constrains copper coordination in blue proteins.

In azurins and other blue copper proteins with relatively low reduction potentials (E(0) [Cu(II)/Cu(I)]<400 mV vs. normal hydrogen electrode), the folded polypeptide framework constrains both copper(II) and copper(I) in such a way as to tune the reduction potentials to values that differ greatly from those for most copper complexes. Largely conserved networks of hydrogen bonds organize and lock the rest of the folded protein structure to a loop that contains three of the ligands to copper. Changes in hydrogen bonds that allow copper(I) to revert more closely to its preferred geometry [relative to the copper(II) geometry] accordingly lead to an increase in E(0). This paper reports mutations in the ligand loop of amicyanin from P. denitrificans that relax the constraints on ligation for copper(I) and significantly raise E(0) for these mutants (for example 415+/-4 mV) relative to that of the native amicyanin (265+/-4 mV). These mutations also shift the pK(a) of a ligand histidine to below 5 relative to 7.0 in the wild type.