Strong modulation of nitrite reductase activity of cytoglobin by disulfide bond oxidation: Implications for nitric oxide homeostasis

Globin-mediated nitric oxide (NO) dioxygenase and nitrite reductase activities have been proposed to serve protective functions within the cell by scavenging or generating NO respectively. Cytoglobin has rapid NO dioxygenase activity, similar to other globins, however, the apparent rates of nitrite reductase activity have been reported as slow or negligible. Here we report that the activity of cytoglobin nitrite reductase activity is strongly dependent on the oxidation state of the two surface-exposed cysteine residues. The formation of an intramolecular disulfide bond between cysteines C38 and C83 enhances the nitrite reductase activity by 50-fold over that of the monomer with free sulfhydryl or 140-fold over that of the dimer with intermolecular disulfide bonds. The NO dioxygenase reactivity of cytoglobin is very rapid with or without disulfide bond, however, binding of the distal histidine following dissociation of the nitrate are affected by the presence or absence of the disulfide bond. The nitrite reductase activity reported here for the monomer with intramolecular disulfide is much higher than of those previously reported for other mammalian globins, suggesting a plausible role for this biochemistry in controlling NO homeostasis the cell under oxidative and ischemic conditions

Win Some, Lose Some

Photograph used for a story in the Daily Oklahoman newspaper. Caption: "Nevada-Las Vegas head coach Jerry Tarkanian, left, gives a dismal look during his team's game against Kansas, where UNLV lost in the National Invitational Tournament in New York Wednesday. (Kansas 91, UNLV 77)

Win Some, Lose Some

Photograph used for a story in the Daily Oklahoman newspaper. Caption: "Nevada-Las Vegas head coach Jerry Tarkanian, left, gives a dismal look during his team's game against Kansas, where UNLV lost in the National Invitational Tournament in New York Wednesday. (Kansas 91, UNLV 77)

Hemoglobin conformation couples erythrocyte S-nitrosothiol content to O(2) gradients

It is proposed that the bond between nitric oxide (NO) and the Hb thiol Cys-β(93) (SNOHb) is favored when hemoglobin (Hb) is in the relaxed (R, oxygenated) conformation, and that deoxygenation to tense (T) state destabilizes the SNOHb bond, allowing transfer of NO from Hb to form other (vasoactive) S-nitrosothiols (SNOs). However, it has not previously been possible to measure SNOHb without extensive Hb preparation, altering its allostery and SNO distribution. Here, we have validated an assay for SNOHb that uses carbon monoxide (CO) and cuprous chloride (CuCl)-saturated Cys. This assay is specific for SNOs and sensitive to 2–5 pmol. Uniquely, it measures the total SNO content of unmodified erythrocytes (RBCs) (SNO(RBC)), preserving Hb allostery. In room air, the ratio of SNO(RBC) to Hb in intact RBCs is stable over time, but there is a logarithmic loss of SNO(RBC) with oxyHb desaturation (slope, 0.043). This decay is accelerated by extraerythrocytic thiol (slope, 0.089; P < 0.001). SNO(RBC) stability is uncoupled from O(2) tension when Hb is locked in the R state by CO pretreatment. Also, SNO(RBC) is increased ≈20-fold in human septic shock (P = 0.002) and the O(2)-dependent vasoactivity of RBCs is affected profoundly by SNO content in a murine lung bioassay. These data demonstrate that SNO content and O(2) saturation are tightly coupled in intact RBCs and that this coupling is likely to be of pathophysiological significance

Endogenous S-nitrosothiols protect against myocardial injury

Despite substantial evidence that nitric oxide (NO) and/or endogenous S-nitrosothiols (SNOs) exert protective effects in a variety of cardiovascular diseases, the molecular details are largely unknown. Here we show that following left coronary artery ligation, mice with a targeted deletion of the S-nitrosoglutathione reductase gene (GSNOR−/−) have reduced myocardial infarct size, preserved ventricular systolic and diastolic function, and maintained tissue oxygenation. These profound physiological effects are associated with increases in myocardial capillary density and S-nitrosylation of the transcription factor hypoxia inducible factor-1α (HIF-1α) under normoxic conditions. We further show that S-nitrosylated HIF-1α binds to the vascular endothelial growth factor (VEGF) gene, thus identifying a role for GSNO in angiogenesis and myocardial protection. These results suggest innovative approaches to modulate angiogenesis and preserve cardiac function