Characterization of NADPH-dependent methemoglobin reductase as a heme-binding protein present in erythrocytes and liver.

Abstract

An NADPH-dependent reductase, first shown in the 1930s to catalyze the methylene blue-dependent reduction of methemoglobin in erythrocytes, has now been characterized as a high-affinity heme-binding protein and has been detected in liver. Highly purified bovine erythrocyte reductase binds protohemin to form a 1:1 complex with a Kd of 7 nM. Binding of protohemin completely inhibits reductase activity. Other tetrapyrroles and fatty acids also bind to the reductase and inhibit its activity. Protoporphyrin, hematoporphyrin, and coproporphyrin form 1:1 complexes withKd values ranging from 1 to 5 IzM. The inhibition constants for a number of saturated and unsaturated fatty acids range from 6 to 52 ,LM. A protein that is immunologically cross-reactive to the reductase has been detected in the cytosolic fractions of bovine and rat liver and of bovine, rat, rabbit, and human erythrocytes. By immunoblot analysis, the bovine liver and erythrocyte proteins appear identical in size, as do the rat liver and erythrocyte proteins. The concentration of the protein in bovine erythrocytes has been estimated by quantitative immunoblotting to be 10 juM. The detection of this protein in liver cells, the demonstration of its binding properties, and its weak reductase activity bring into question the long-held belief that this is uniquely an erythrocyte protein and that it functions as a reductase.Methylene blue stimulation of methemoglobin reduction in human erythrocytes was described 60 years ago by Warburg et al. (1), Steele and Spink (2), and Williams and Challis (3). Subsequent work by a number of investigators resulted in preparations of a cytosolic NADPH-dependent reductase that catalyzed the reduction of methylene blue and flavins and, in the presence of redox couplers, catalyzed the reduction of methemoglobin (4-9). Whereas this NADPHdependent reductase is believed to contribute very little to methemoglobin reduction under normal conditions (10), its catalysis of methemoglobin reduction in the presence of methylene blue or riboflavin is the basis for the use of these compounds as therapeutic agents in the treatment of congenital and toxic methemoglobinemia (11)(12)(13). The erythrocyte reductase has variously been referred to as NADPH dehydrogenase, diaphorase, methemoglobin reductase, and, most recently, flavin reductase.The reductase has been shown to bind small molecules. Isolation procedures generally yield two forms of the reductase, which exhibit very similar activities but different isoelectric points; one, but not the other, of these forms is reported to possess protein-bound NADP+ and a proteinbound yellow chromophore of unknown structure (7,8,(14)(15)(16). The isolation of the protein in the presence of flavin yields a flavoprotein (17). The protein isolated without the addition of flavin contains no flavin and yet exhibits full reductase activity.Recently (18) Bovine erythrocyte green heme-binding protein was purified as described (19). Antibodies to the green heme-binding protein were raised in young adult ...

Studies on the Specificity of the Tetrapyrrole Substrate for Human Biliverdin-IXα Reductase and Biliverdin-IXβ Reductase

“…The inhibition is competitive with respect to FMN and mixed against NADPH, consistent with the hypothesis that the verdin and flavin may compete for a common site (45). Protohemin, which has been reported previously to bind tightly to bovine liver flavin reductase (46), also acts as a potent inhibitor of the human enzyme giving an IC 50 of 0.8 M under standard assay conditions. This is also consistent with the hypothesis of a common tetrapyrrole/flavin site.…”

“…The number of compounds known to interact with BVR-B/FR now includes a wide range of biliverdin isomers in addition to pyrroloquinoline quinone (48), various hemes, fatty acids, and porphyrins (46).…”

BLVRB redox mutation defines heme degradation in a metabolic pathway of enhanced thrombopoiesis in humans

“…28 Interestingly, BV isomer generation appears distinct in the fetus where BV IXb may predominate, 41 although the mechanism for BV IXb generation remains enigmatic. Nonetheless, BLVRB binds promiscuously (and nonproductively) to other tetrapyrroles such as protohemin 27 or BV IXa, 28 suggesting that its redox activity could be modulated by endogenous cellular heme byproducts with resultant effects on redox coupling and lineage commitment (ie, function as a metabolically regulated cellular rheostat 34 ). The loss-of-function redox mutation causes defective ROS handling in the background of endogenous BLVRB expression, establishing a dominant inhibitory mechanism of action.…”

Functional Properties of Neuroglobin and Cytoglobin. Insights into the Ancestral Physiological Roles of Globins

“…The Fe 2+ 7O 2 complex of Ngb is unstable in vitro, whereby formation of ferric heme upon air exposure at room temperature occurs within a few minutes (7,19). Ngb-mediated reversible O 2 binding within neurons can only be possible if an enzymatic met-globin reducing system is present, similar to the met-Hb reductase system identified within red blood cells (23) or to that used in in vitro O 2 equilibrium measurements (19,24). However, it appears unlikely that Ngb, which has retained a relatively conserved primary structure during evolution, should be able to bind O 2 reversibly only by means of a metabolically-expensive consumption of reducing equivalents.…”