Three mammalian cytochromes b₅₆₁ are ascorbate‐dependent ferrireductases

Abstract

Cytochromes b561 are a family of transmembrane proteins found in most eukaryotic cells. Three evolutionarily closely related mammalian cytochromes b561 (chromaffin granule cytochrome b, duodenal cytochrome b, and lysosomal cytochrome b) were expressed in a Saccharomyces cerevisiaeΔfre1Δfre2 mutant, which lacks almost all of its plasma membrane ferrireductase activity, to study their ability to reduce ferric iron (Fe3+). The expression of each of these cytochromes b561 was able to rescue the growth defect of the Δfre1Δfre2 mutant cells in iron‐deficient conditions, suggesting their involvement in iron metabolism. Plasma membrane ferrireductase activities were measured using intact yeast cells. Each cytochrome b561 showed significant FeCN and Fe3+‐EDTA reductase activities that were dependent on the presence of intracellular ascorbate. Site‐directed mutagenesis of lysosomal cytochrome b was conducted to identify amino acids that are indispensable for its activity. Among more than 20 conserved or partially conserved amino acids that were investigated, mutations of four His residues (H47, H83, H117 and H156), one Tyr (Y66) and one Arg (R67) completely abrogated the FeCN reductase activity, whereas mutations of Arg (R149), Phe (F44), Ser (S115), Trp (W119), Glu (E196), and Gln (Q131) affected the ferrireductase activity to some degree. These mutations may affect the heme coordination, ascorbate binding, and/or ferric substrate binding. Possible roles of these residues in lysosomal cytochrome b are discussed. This study demonstrates the ascorbate‐dependent transmembrane ferrireductase activities of members of the mammalian cytochrome b561 family of proteins.

Human Erythrocyte Membranes Contain a Cytochrome b561 That May Be Involved in Extracellular Ascorbate Recycling

“…The only proposed function for DCytb so far is ferrireductase activity (25,27). However, it is possible that DCytb has the same function as CGCytb in chromaffin granules in that it can recycle Asc by reducing MDHA outside of erythrocytes.…”

“…However, it is possible that DCytb has the same function as CGCytb in chromaffin granules in that it can recycle Asc by reducing MDHA outside of erythrocytes. Indeed, in addition to its well documented function as an MDHA reductase, CGCytb also showed ferrireductase activity when expressed in yeast (27) and Xenopus oocytes (28). It is possible that these Cyts b 561 can mediate both activities, with one activity favored depending on the cellular milieu.…”

“…The most likely function for this activity in the developing erythroblast is to reduce ferric iron during its uptake and use in hemoglobin synthesis. Because DCytb shows Asc-dependent FeCN reductase activity when expressed in yeast (27), it is possible that it includes part of the erythroblast iron reduction/uptake system. However, all components of the hemoglobin-synthesizing system, as well as the transferrin receptor and the ability to take up iron, are lost during the maturation of reticulocytes (52)(53)(54)(55)(56).…”

“…Genetic and biochemical studies support a role for DCytb in iron metabolism, where it functions as a ferrireductase (25). Although the MDHA reductase activity of CGCytb has been documented for many years, we and others have shown that this protein also displays ferrireductase activity when expressed in yeast (27) or Xenopus oocytes (28). It therefore remains an open question whether these Cyts b 561 can have both MDHA reductase and ferrireductase activities in vivo or whether one of these activities is favored under physiological conditions.…”

Structure and mechanism of a eukaryotic transmembrane ascorbate-dependent oxidoreductase

“…Ascorbate serves as an electron donor for various enzymes, such as prolyl and lysyl hydroxylase, dopamine β-hydroxylase, ascorbate peroxidase, and cytochrome b 561 (Cyt b 561 ). Cyt b 561 , initially identified in the chromaffin granules of bovine adrenal medullae about 40 y ago (7,8), is a transmembrane ascorbate-dependent oxidoreductase (9)(10)(11)(12)(13) that plays a key role in ascorbate recycling and other physiological processes, such as iron absorption (14). To our knowledge, Cyt b 561 is the only membrane-embedded oxidoreductase that relies on ascorbate as the electron donor.…”