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The cGMP-dependent protein kinase G-1α (PKG-1α) is a downstream mediator of nitric oxide and natriuretic peptide signaling. Alterations in this pathway play a key role in the pathogenesis and progression of vascular diseases associated with increased vascular tone and thickness, such as pulmonary hypertension. Previous studies have shown that tyrosine nitration attenuates PKG-1α activity. However, little is known about the mechanisms involved in this event. Utilizing mass spectrometry, we found that PKG-1α is susceptible to nitration at tyrosine 247 and 425. Tyrosine to phenylalanine mutants, Y247F- and Y425F-PKG-1α, were both less susceptible to nitration than WT PKG-1α, but only Y247F-PKG-1α exhibited preserved activity, suggesting that the nitration of Tyr(247) is critical in attenuating PKG-1α activity. The overexpression of WT- or Y247F-PKG-1α decreased the proliferation of pulmonary artery smooth muscle cells (SMC), increased the expression of SMC contractile markers, and decreased the expression of proliferative markers. Nitrosative stress induced a switch from a contractile to a synthetic phenotype in cells expressing WT- but not Y247F-PKG-1α. An antibody generated against 3-NT-Y247 identified increased levels of nitrated PKG-1α in humans with pulmonary hypertension. Finally, to gain a more mechanistic understanding of how nitration attenuates PKG activity, we developed a homology model of PKG-1α. This model predicted that the nitration of Tyr(247) would decrease the affinity of PKG-1α for cGMP, which we confirmed using a [(3)H]cGMP binding assay. Our study shows that the nitration of Tyr(247) and the attenuation of cGMP binding is an important mechanism regulating in PKG-1α activity and SMC proliferation/differentiation.

Photoinhibition and production of reactive oxygen species were studied in tobacco plants overexpressing the plastid terminal oxidase (PTOX). In high light, these plants was more susceptible to photoinhibition than wild-type plants. Also oxygen-evolving activity of isolated thylakoid membranes from the PTOX-overexpressing plants was more strongly inhibited in high light than in thylakoids from wild-type plants. In contrast in low light, in the PTOX overexpressor, the thylakoids were protected against photoinhibition while in wild type they were significantly damaged. The production of superoxide and hydroxyl radicals was shown by EPR spin-trapping techniques in the different samples. Superoxide and hydroxyl radical production was stimulated in the overexpressor. Two-thirds of the superoxide production was maintained in the presence of DNP-INT, an inhibitor of the cytochrome b(6)f complex. No increase of the SOD content was observed in the overexpressor compared with the wild type. We propose that superoxide is produced by PTOX in a side reaction and that PTOX can only act as a safety valve under stress conditions when the generated superoxide is detoxified by an efficient antioxidant system.

We report the characterization of the effects of the A249S mutation located within the binding pocket of the primary quinone electron acceptor, Q(A), in the D2 subunit of photosystem II in Thermosynechococcus elongatus. This mutation shifts the redox potential of Q(A) by approximately -60 mV. This mutant provides an opportunity to test the hypothesis, proposed earlier from herbicide-induced redox effects, that photoinhibition (light-induced damage of the photosynthetic apparatus) is modulated by the potential of Q(A). Thus the influence of the redox potential of Q(A) on photoinhibition was investigated in vivo and in vitro. Compared with the wild-type, the A249S mutant showed an accelerated photoinhibition and an increase in singlet oxygen production. Measurements of thermoluminescence and of the fluorescence yield decay kinetics indicated that the charge-separated state involving Q(A) was destabilized in the A249S mutant. These findings support the hypothesis that a decrease in the redox potential of Q(A) causes an increase in singlet oxygen-mediated photoinhibition by favoring the back-reaction route that involves formation of the reaction center chlorophyll triplet. The kinetics of charge recombination are interpreted in terms of a dynamic structural heterogeneity in photosystem II that results in high and low potential forms of Q(A). The effect of the A249S mutation seems to reflect a shift in the structural equilibrium favoring the low potential form.