Modulation of the flavin-protein interactions in NADH peroxidase and mercuric ion reductase: a resonance Raman study.

NADH peroxidase (Npx) and mercuric ion reductase (MerA) are flavoproteins belonging to the pyridine nucleotide:disulfide oxidoreductases (PNDO) and catalyzing the reduction of toxic substrates, i.e., hydrogen peroxide and mercuric ion, respectively. To determine the role of the flavin adenine dinucleotide (FAD) in the detoxification mechanism, the resonance Raman (RR) spectra of these enzymes under various redox and ligation states have been investigated using blue and/or near-UV excitation(s). These data were compared to those previously obtained for glutathione reductase (GR), another enzyme of the PNDO family, but catalyzing the reduction of oxidized glutathione. Spectral differences have been detected for the marker bands of the isoalloxazine ring of Npx, MerA, and GR. They provide evidence for different catalytic mechanisms in these flavoproteins. The RR modes of the oxidized and two-electron reduced (EH) forms of Npx are related to very tight flavin-protein interactions maintaining a nearly planar conformation of the isoalloxazine tricycle, a low level of H-bonding at the N/N and O/O sites, and a strong H-bond at NH. They also indicate minimal changes in FAD structure and environment upon either NAD(H) binding or reduction of the sulfinic redox center. All these spectroscopic data support an enzyme functioning centered on the Cys-SO/Cys-S redox moiety and a neighbouring His residue. On the contrary, the RR data on various functional forms of MerA are indicative of a modulation of both ring II distortion and H-bonding states of the N site and ring III. The Cd(II) binding to the EH-NADP(H) complexes, biomimetic intermediates in the reaction of Hg(II) reduction, provokes important spectral changes. They are interpreted in terms of flattening of the isoalloxazine ring and large decreases in H-bonding at the N site and ring III. The large flexibility of the FAD structure and environment in MerA is in agreement with proposed mechanisms involving C(flavin) adducts.