Nearly 35 years ago, Hodgson and Fridovich proposed that the reaction between H2
and SOD could generate a copper-bound hydroxyl radical (SOD-Cu(II)-•
OH) that reacts with HCO3−
to form a diffusible oxidant, CO3−
that oxidized several peroxidatic substrates (ABTS, dichlorodihydrofluorescein, and others) outside of the active site (4
). More recently, it was shown that CO2
, not HCO3−
, undergoes peroxidation to CO3−
in the presence of H2
and SOD (29
). Using EPR spin-trapping methods, evidence for CO3−
and other radicals derived from it was demonstrated during HCO3−
-stimulated peroxidase activity of SOD (11
). The oxidant derived from HCO3−
(i.e., carbonate anion radical) was proposed to react with surface-associated Trp-32 in human SOD forming a tryptophanyl radical (31
). Mutation of Trp-32 by phenylalanine totally eliminated the Trp-32 radical formed from hSOD reaction with H2
), providing additional evidence for the reaction between CO3−
and tryptophan-32 on the surface of the protein (31
An alternative mechanism for HCO3−
-mediated peroxidase activity has also been proposed. It was proposed that the active species (peroxycarbonate or HCO4−
) formed during the peroxidase activity is enzyme-associated and non-diffusible (32
). It was also suggested that the enzyme-associated oxidant (HCO4−
, a non-radical) does not diffuse away from the active site but reacts locally at the active site of copper-bound histidines. The present magnetic resonance analyses clearly rule out the “enzyme-bound peroxycarbonate” as an oxidant responsible for oxidation of the “distant” histidine residues. The mechanism of direct oxidation of histidine at the active site by peroxycarbonate remains to be determined, however.
It was shown that SOD has a thiol oxidase activity (16
). Previously, we proposed that HCO3−
-stimulated peroxidase activity further accelerated thiol oxidase activity (24
). Using a kinetic simulation model, the EPR profile changes in SOD-Cu(II) were simulated (24
). Thiol oxidase activity generated in situ
needed for SOD peroxidase activity that was further stimulated by bicarbonate. The peroxidase activity enhanced thiol depletion and oxygen consumption resulting in increased thiol oxidase activity via
formation of a diffusible CO3−
The ESEEM experiment offers a sensitive means for viewing the structural relationship between Cu(II) and its four coordinated histidine ligands (25
). In a HYSCORE experiment, the hyperfine coupling between Cu(II) and the remote nitrogen of a strongly bound histidyl imidazole ligand gives rise to cross peaks near (1.5, 4.0 MHz) whose contours are parallel to the frequency axes. The HYSCORE spectrum of SOD at g = 2.06 () shows two of these double quantum, dq-dq, cross peaks of nearly equal intensity that we can assign to a stronger set of hyperfine couplings due to H44 and H46, and a weaker set of couplings arising from H61 and H118 based on previous ESEEM studies (27
). Treatment of the enzyme with H2
causes the ESEEM depth or amplitude to decrease by about 40% and the dq-dq correlation in the HYSCORE spectrum () is altered to show a more intense broader correlation centered at (1.4, 4.0 MHz) and a minor contribution at (1.6, 4.4 MHz). Previous studies of SOD treated with peroxide have shown that H118 is selectively oxidized to form a 2-oxo-histidine species (9
). Although the 14
N-ESEEM amplitude is a function of the interplay between ligand hyperfine, nuclear Zeeman and nuclear quadrupole interactions, the HYSCORE spectra show that the hyperfine coupling changes that result from peroxide treatment are minor. The 40% loss in ESEEM amplitude can only be explained by the loss of at least one Cu(II)-histidine hyperfine interaction, commensurate with the breaking of the Cu(II) - H118 bond. This bond-breaking chemistry then leads to the changes in hyperfine coupling for the remaining histidyl imidazole ligands that are captured in the HYSCORE cross peak pattern centered at (1.4, 4.0 MHz). The previous biochemical studies that showed selective oxidation of H118, also showed that this modification only occurred in 66% of the Cu(II) sites. The residual cross peak due to the stronger Cu(II)-histidine interaction in H2
treated SOD () likely represents the fraction of Cu(II) sites where H118 is not oxidized and remains bound to Cu(II). Finally, the ESEEM data show conclusively that peroxide treatment in the presence of physiologically relevant levels of bicarbonate does not alter the ligation of H44, H46, H61 and H118 to Cu(II).
The present NMR study has limitations in that the 2D homonuclear NMR experiments using the commercially available protein enabled only the histidine analysis. The best way to confirm the structural assignments would have been to make recombinant SOD and the histidine mutants. To characterize the specific histidine modification, 15N-labeled recombinant SOD should be used. Lack of these studies clearly present a limited scope for detailed experimental interpretations on the oxidative modification.
The present data using the combined approach involving ESEEM/HYSCORE, ENDOR, and 1D-NMR techniques are consistent with the previous mass spectral studies (9
) indicating His118 oxidation during inactivation of Cu, Zn SOD with H2
. The previous results also reported that histidine oxidation at other copper sites might be involved (9
). The present magnetic resonance analyses also suggest marked changes in the hyperfine couplings at other copper(II)-histidine sites. These results reveal that the oxidation of His residues in Cu, Zn, SOD in the presence of H2
alone is presumably more extensive. However, the NMR and ESEEM/HYSCORE results indicate that in the presence of bicarbonate the histidine oxidation is selective, occurring outside of the active site.
The present results are significant because HCO3− is abundant in all living cells protecting SOD from its oxidative damage at the active site but causes extensive damage to outer residues of SOD or other vital proteins as observed in neurodegenerative diseases. A large body of evidence indicates that elevated oxidative stress perhaps due to peroxidase/thiol oxidase stimulated peroxidase activity of SOD could play a major role in free radical biology. Finally, the combined use of NMR and EPR techniques is a powerful approach to elucidate the structural biological changes induced by site-specific generation of oxidants in biomolecules.
The combined NMR and pulse EPR data show that in the absence of HCO3−, the Cu(II) binding histidine residues were specifically oxidized and the other histidine residues were not affected during peroxidase and thiol oxidase activity of SOD. However, in the presence of HCO3−, the Cu(II) bound histidines in SOD were unaffected; instead, a distant histidine residue () was oxidized by a diffusible oxidant (most likely CO3−) formed at the active site of SOD.