Abstract
As part of our studies on the nitric oxide-related pathology of cerebral malaria, we show that the antioxidative enzyme glutathione reductase (GR) is inactivated by peroxynitrite, with GR from the malarial parasite Plasmodium falciparum being more sensitive than human GR. The crystal structure of modified human GR at 1.9-A resolution provides the first picture of protein inactivation by peroxynitrite and reveals that this is due to the exclusive nitration of 2 Tyr residues (residues 106 and 114) at the glutathione disulfide-binding site. The selective nitration explains the impairment of binding the peptide substrate and thus the nearly 1000-fold decrease in catalytic efficiency (k(cat)/K(m)) of glutathione reductase observed at physiologic pH. By oxidizing the catalytic dithiol to a disulfide, peroxynitrite itself can act as a substrate of unmodified and bisnitrated P. falciparum glutathione reductase.
Highlights
Nitric oxide (NO) is a pluripotent molecule that is involved in both cytoprotective and cytotoxic processes [1,2,3,4]
As part of our studies on the nitric oxide-related pathology of cerebral malaria, we show that the antioxidative enzyme glutathione reductase (GR) is inactivated by peroxynitrite, with GR from the malarial parasite Plasmodium falciparum being more sensitive than human GR
We show that PfGR is an effective catalyst of ONOOH-dependent oxidation of NADPH
Summary
Materials—Peroxynitrite was synthesized from sodium nitrite and hydrogen peroxide using established protocols [27, 28] and was stored at Ϫ80 °C. A more careful examination of the 2Fo Ϫ Fc electron density map suggested a mixture of wild-type Tyr106 and nitrated Tyr106 This was further confirmed when preliminary positional and individual B-factor refinements with nitrated Tyr106 at full occupancy produced an average B-factor of ϳ30 Å2 for phenol ring atoms and of ϳ50 Å2 for atoms of the 3-nitro group. To estimate the nitrated Tyr106/wild-type Tyr106 ratio, test refinements consisting of 30 cycles of conventional positional and 30 cycles of restrained B-factor refinements were carried out with nitrated Tyr106 at occupancies of 0.5, 0.6, 0.7, and 0.8, whereas the native conformation of Tyr106 was maintained as an alternate conformation at a complementary occupancy The goal of this approach was (i) to find those combinations of occupancies that would produce near wild-type B-values for the wild-type conformation (ϳ20 Å2), and uniform B-values for all atoms in all other conformations and (ii) to minimize peaks in Fo Ϫ Fc electron density maps. We estimate that the limit of sensitivity of the difference Fourier is an occupancy of ϳ20%
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