Enzymatic oxidation of two phenolic compounds [syringic acid (3,5-dimethoxy-4-hydroxybenzoic acid) and 2,6-dimethylphenol] was studied. The products of laccase- and laccase-mediator-catalyzed oxidation reactions were monitored by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and further analyzed by electrospray ionization Fourier transform ion cyclotron resonance (ESI-FTICR) MS with collision-induced dissociation (CID) experiments. For the oligomers of syringic acid, some variability was observed in MALDI-TOF analysis. However, the origin of this variability could not be resolved on the basis of MALDI-TOF spectra due to the poor resolution of the instrument in use. The strength of ESI-FTICR MS was the high-resolution data provided from oligomers of syringic acid. The CID experiments were extremely useful for structural studies of oligomers and verified that the variability of the products was due to the end groups; the phenolic hydroxyl group was modified during the oxidation.

Substituted benzyl alcohol was oxidized enzymatically with a laccase-mediator system and the products were investigated as a function of time by nanoelectrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (nanoESI-FTICRMS). With Trametes versicolor laccase (TVL), the mediator, 2,2',6,6'-tetramethylpiperidine-N-oxyl radical (TEMPO), undergoes oxidation and forms oxoammonium ion. Oxidized TEMPO oxidizes the alcohol and is simultaneously reduced to the N-OH form. The laccase then restores TEMPO back to the normal radical form and the oxidation cycle starts again. The role of TEMPO and the structures of its oxidized and reduced forms in the enzymatic oxidation process were clarified in collision-induced dissociation experiments and gas-phase hydrogen/deuterium (H/D) exchange reactions. The amounts of enzyme and mediator were significant for product formation: with greater amounts overoxidation products, the corresponding benzoic acid and benzonitrile were formed. Smaller amounts of laccase and mediator generated benzaldehyde in high yield. The reaction pathway for benzonitrile formation is discussed and it is suggested to start from benzaldehyde and the ammonia in the ammonium acetate buffer.

The oxidation reaction of a laccase mediator ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) was studied by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICRMS). Oxidation products of ABTS were measured after reaction times that varied from a few minutes up to several days and both positive and negative ionization modes were employed. Exact mass measurements and collision-induced dissociation (CID) experiments were used to characterize the structures of the ions formed. After reacting with Trametes versicolor laccase (TvL), the radical cation form of ABTS was the main product observed by the positive ionization mode. Negative ionization mode experiments revealed that a degradation product from ABTS was formed.

The molecular properties and stability of a laccase from the white-rot fungus Trametes hirsuta (ThL) were studied to exploit the unique capability of electrospray ionization mass spectrometry (ESI-MS) to monitor conformational and molecular-based heterogeneities and metal ion binding simultaneously. Acid and organic solvents were applied as denaturing agents. In aqueous acidic solution, ThL existed in two major forms, distinguished by their mass difference; in addition to these, two other forms were detected. This molecular heterogeneity was due to the variable glycan content of ThL. Additionally, copper-depleted forms of laccase were observed in mass spectra measured from aqueous acidic solution. A small amount of organic solvent (acetonitrile, CH(3)CN) increased the loss of one Cu atom from folded states and led to unfolding. In the unfolded state, ThL was depleted of all four copper atoms, and the charge state distribution was shifted to lower mass-to-charge region. Thus, denaturation took place in two stages: first, the loss of one Cu resulting in an inactive form; second, complete denaturation with the loss of the three remaining Cu atoms. After all coppers were lost, ThL was unfolded, as was clearly seen in the increased number of charge states in the mass spectra. Different stabilities of the glycoforms were observed in the denaturation triggered in acid and organic solvents.