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• W2060193350 abstract "A plasma membrane iron reductase, required for cellular iron acquisition by Saccharomyces cerevisiae, and the human phagocytic NADPH oxidase, implicated in cellular defense, contain low potential plasma membrane b cytochromes that share elements of structure and function. Four critical histidine residues in the FRE1 protein of the iron reductase were identified by site-directed mutagenesis. Individual mutation of each histidine to alanine eliminated the entire heme spectrum without affecting expression of the apoprotein, documenting the specificity of the requirement for the histidine residues. These critical residues are predicted to coordinate a bis-heme structure between transmembrane domains of the FRE1 protein. The histidine residues are conserved in the related gp91phox protein of the NADPH oxidase of human granulocytes, predicting the sites of heme coordination in that protein complex. Similarly spaced histidine residues have also been implicated in heme binding by organelle b cytochromes with little overall sequence similarity to the plasma membrane b cytochromes. This bis-heme motif may play a role in transmembrane electron transport by distinct families of polytopic b cytochromes. A plasma membrane iron reductase, required for cellular iron acquisition by Saccharomyces cerevisiae, and the human phagocytic NADPH oxidase, implicated in cellular defense, contain low potential plasma membrane b cytochromes that share elements of structure and function. Four critical histidine residues in the FRE1 protein of the iron reductase were identified by site-directed mutagenesis. Individual mutation of each histidine to alanine eliminated the entire heme spectrum without affecting expression of the apoprotein, documenting the specificity of the requirement for the histidine residues. These critical residues are predicted to coordinate a bis-heme structure between transmembrane domains of the FRE1 protein. The histidine residues are conserved in the related gp91phox protein of the NADPH oxidase of human granulocytes, predicting the sites of heme coordination in that protein complex. Similarly spaced histidine residues have also been implicated in heme binding by organelle b cytochromes with little overall sequence similarity to the plasma membrane b cytochromes. This bis-heme motif may play a role in transmembrane electron transport by distinct families of polytopic b cytochromes. INTRODUCTIONThe human phagocyte NADPH oxidase is a heme-containing enzyme complex critical for defense against microorganisms (1Thrasher A.J. Keep N.H. Wientjes F. Segal A.W. Biochim. Biophys. Acta. 1994; 1227: 1-24Crossref PubMed Scopus (212) Google Scholar). Defective functioning of this oxidase results in the susceptibility to infection that characterizes patients with chronic granulomatous disease. The genetic defects affecting the oxidase are currently the focus of corrective gene therapy trials (2Thrasher A.J. de Alwis M. Casimir C.M. Kinnon C. Page K. Lebkowski J. Segal A.W. Levinsky R.J. Blood. 1995; 86: 761-765Crossref PubMed Google Scholar, 3Li F. Linton G.F. Sekhsaria S. Whiting-Theobald N. Katkin J.P. Gallin J.I. Malech H.L. Blood. 1994; 84: 53-58Crossref PubMed Google Scholar). The NADPH oxidase assembles from two membrane (gp91phox and p22phox which together comprise flavocytochrome b558) and three cytosolic (p67phox, p47phox, and p40phox) components in response to a signaling pathway mediated through the small GTP-binding protein Rac (1Thrasher A.J. Keep N.H. Wientjes F. Segal A.W. Biochim. Biophys. Acta. 1994; 1227: 1-24Crossref PubMed Scopus (212) Google Scholar). The heme cofactor is required for enzymatic function: the transfer of electrons from cytosolic NADPH across the vacuolar membrane to molecular oxygen. The superoxide generated by this process in turn gives rise to toxic byproducts which are used in cellular defense against ingested bacteria and fungi (1Thrasher A.J. Keep N.H. Wientjes F. Segal A.W. Biochim. Biophys. Acta. 1994; 1227: 1-24Crossref PubMed Scopus (212) Google Scholar).Despite efforts by several laboratories, the sites within the oxidase where the essential heme cofactors are bound have not been defined (4Quinn M.T. Mullen J.L. Jesaitis A.J. J. Biol. Chem. 1992; 267: 7303-7309Abstract Full Text PDF PubMed Google Scholar, 5Cross A.R. Rae J. Curnutte J.T. J. Biol. Chem. 1995; 270: 17075-17077Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 6Segal A.W. Immunodeficiency. 1993; 4: 167-179PubMed Google Scholar). Heme copurifies with the cytochrome b subunits, gp91phox and p22phox (6Segal A.W. Immunodeficiency. 1993; 4: 167-179PubMed Google Scholar). However, inherited mutations in either of these subunits are associated with the absence of detectable heme in the enzyme complex and destabilization of the proteins, thus frustrating efforts to identify the specific heme-liganding residues (7Roos D. de Boer M. Kuribayashi F. Meischl C. Weening R.S. Segal A.W. Ahlin A. Nemet K. Hossle J.P. Bernatowska-Matuszkiewicz E. Middleton-Price H. Blood. 1996; 87: 1663-1681Crossref PubMed Google Scholar).We undertook to address this problem via study of the FRE1 reductase of the yeast Saccharomyces cerevisiae. FRE1 is homologous to gp91phox (8Dancis A. Roman D.G. Anderson G.J. Hinnebusch A.G. Klausner R.D. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 3869-3873Crossref PubMed Scopus (281) Google Scholar) and encodes a plasma membrane-associated subunit of an iron reductase (9Lesuisse E. Casteras-Simon M. Labbe P. J. Biol. Chem. 1996; 271: 13578-13583Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). FRE1 or FRE2 is required for iron uptake in yeast (10Georgatsou E. Alexandraki D. Mol. Cell. Biol. 1994; 14: 3065-3073Crossref PubMed Scopus (197) Google Scholar). FRE1 is required for the major (90%) extracellular ferric reductase activity under most conditions and appears to be a structural subunit of the externally directed reductase activity (8Dancis A. Roman D.G. Anderson G.J. Hinnebusch A.G. Klausner R.D. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 3869-3873Crossref PubMed Scopus (281) Google Scholar). Chelated ferric iron outside the cell is reduced by this activity to ferrous iron and then transported into the cell by the high-affinity iron uptake system (11Stearman R. Yuan D.S. Yamaguchi-Iwai Y. Klausner R.D. Dancis A. Science. 1996; 271: 1552-1557Crossref PubMed Scopus (574) Google Scholar). Thus, this iron reductase of yeast, like the NADPH oxidase of humans, is involved in the transfer of electrons from a cytosolic donor across a membrane to an extracellular acceptor.The yeast iron reductase, like the human NADPH oxidase, requires heme for its function. The requirement of heme for the plasma membrane ferric reductase of yeast is supported by the lack of activity in mutants deficient in heme biosynthesis (12Lesuisse E. Labbe P. J. Gen. Microbiol. 1989; 135: 257-263PubMed Google Scholar). In addition, FRE1 expression levels correlate with the visible spectrum attributed to the b heme cofactor (13Shatwell K.P. Dancis A. Cross A.R. Klausner R.D. Segal A.W. J. Biol. Chem. 1996; 271: 14240-14244Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). The appearance of this visible spectrum is extremely similar to the spectrum of the purified NADPH oxidase b cytochrome (13Shatwell K.P. Dancis A. Cross A.R. Klausner R.D. Segal A.W. J. Biol. Chem. 1996; 271: 14240-14244Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). Two hemes are probably present in each complex of the NADPH oxidase, as indicated by quantitation of heme in the purified cytochrome (4Quinn M.T. Mullen J.L. Jesaitis A.J. J. Biol. Chem. 1992; 267: 7303-7309Abstract Full Text PDF PubMed Google Scholar, 14Segal A.W. West I. Wientjes F. Nugent J.H.A. Chavan A.J. Haley B. Garcia R.C. Rosen H. Scrace G. Biochem. J. 1992; 284: 781-788Crossref PubMed Scopus (289) Google Scholar). Midpoint potential titration of the gp91phox Arg54→ Ser mutant is also consistent with the presence of two hemes with potentials of −220 and −300 mV (5Cross A.R. Rae J. Curnutte J.T. J. Biol. Chem. 1995; 270: 17075-17077Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar), and subsequent reanalysis of the data for the normal protein indicates the presence of two low potential hemes of −225 and −265 mV (5Cross A.R. Rae J. Curnutte J.T. J. Biol. Chem. 1995; 270: 17075-17077Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). Data from resonance Raman spectroscopy suggests a bis-imidazole coordination (15Hurst J.K. Loehr T.M. Curnutte J.T. Rosen H. J. Biol. Chem. 1991; 266: 1627-1634Abstract Full Text PDF PubMed Google Scholar). The low potential hemes distinguish this enzyme from most other heme enzymes. In this regard, it is significant that redox titration of the heme spectrum of the yeast FRE1 reductase also reveals an extremely low midpoint potential of about −250 mV (13Shatwell K.P. Dancis A. Cross A.R. Klausner R.D. Segal A.W. J. Biol. Chem. 1996; 271: 14240-14244Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). The spectral and functional similarities of the yeast FRE1 reductase and the human NADPH oxidase suggested to us that study of the heme ligands in FRE1 might shed light on heme coordination in the human enzyme.RESULTSOur initial objective was to identify the heme-liganding residues within the FRE1 predicted protein. FRE1, like gp91phox, contains multiple hydrophobic domains consistent with transmembrane domains occurring within the amino-terminal two-thirds of the protein. A pair of histidine residues separated by 13 intervening amino acids (His294 and His308) appears within one of these hydrophobic domains. This is followed by an intervening hydrophobic domain and another hydrophobic domain containing a pair of histidines, again separated by 13 amino acids (His364 and His378). The analogous residues in gp91phox can be recognized by their spacing and context (Fig. 1, A and B). A pair of histidine residues separated by 13 amino acids (His101 and His115) appears buried in a hydrophobic region, followed by an intervening hydrophobic domain and another hydrophobic domain containing two histidines separated by 12 amino acids (His209 and His222) (Fig. 1, A and B). A similar motif consisting of two pairs of spaced histidine residues has been linked to heme coordination in organelle b cytochromes of mitochondria (CYTb of the bc1 complex) and chloroplasts (cytb6 of the b6f complex) (21Widger W.R. Cramer W.A. Herrman R.G. Trebst A. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 674-678Crossref PubMed Scopus (334) Google Scholar, 23Cramer W.A. Knaff D.B. Energy Transduction in Biological Membranes: A Textbook of Bioenergetics. Springer-Verlag, New York1990: 330-335Google Scholar, 24Saraste M. FEBS Lett. 1984; 166: 367-372Crossref PubMed Scopus (126) Google Scholar) (Fig. 1A).The contribution of these four conserved histidine residues to the heme binding of FRE1 was therefore examined experimentally. To eliminate background reductase activity, the endogenous FRE1 and FRE2 genes were deleted from the haploid genome of a test strain of yeast (YPH499). Surface reductase activity conferred by transformation of this strain with a high copy-number plasmid carrying FRE1 could then be attributed entirely to expression from the plasmid. Site-directed mutagenesis of the FRE1 plasmid, individually altering histidine residues at amino acid positions 294, 308, 364, or 378 to alanine, reduced the ferric reductase activity of the transformants to levels indistinguishable from the vector control (Fig. 2A). The effect of these mutations upon heme binding was determined by evaluating the reduced-minus-oxidized absorption spectrum of the plasma membranes. Transformants with the mutated FRE1 plasmids almost completely lacked the plasma membrane heme spectrum (Fig. 2B), and the heme content which was calculated from the amplitude of the γ band at 428 nm was negligible (Fig. 2A). The very small amount of residual heme may be attributed to plasma membrane heme proteins other than FRE1 or FRE2 or to very low levels of mitochondrial cross-contamination. As a control, we chose to mutate another histidine in the FRE1 primary sequence. Instead of a random histidine, we selected a residue in a conserved region. This mutated histidine residue (FRE1 amino acid 462) occurs in the conserved HPFT motif (His-Pro-Phe-Tyr) which is situated in the hydrophilic carboxyl-terminal region and is thought to play a role in FAD-isoalloxazine binding (14Segal A.W. West I. Wientjes F. Nugent J.H.A. Chavan A.J. Haley B. Garcia R.C. Rosen H. Scrace G. Biochem. J. 1992; 284: 781-788Crossref PubMed Scopus (289) Google Scholar). The reductase activity of the H462A transformant was 27% compared with the wild-type allele; however, the heme spectrum, although reduced in amplitude, was not fundamentally altered (Fig. 2).Fig. 2FRE1 mutant alleles deficient in reductase activity and heme. A, ferric reductase from whole cells (open bars) and heme levels from plasma membranes (filled bars). Strain 499Δ1Δ2 was transformed with plasmids: lane 1, carrying the wild-type FRE1 allele; lane 2, lacking FRE1; lanes 3-6, containing FRE1 with the critical histidine mutations H294A, H308A, H364A, H378A; or lane 7, containing FRE1 with the HPFT histidine mutation H462A. The transformants were induced and ferric reductase activities were measured (12Lesuisse E. Labbe P. J. Gen. Microbiol. 1989; 135: 257-263PubMed Google Scholar) (open bars). The mean values of three determinations, with standard deviations, are shown. The plasma membranes were purified from cell lysates, and the heme contents were estimated (filled bars). Representative experiments of three determinations are shown. B, spectra of purified plasma membranes. The transformants of strain 499Δ1Δ2 described in A were analyzed by purifying the plasma membranes and resuspending at a concentration of 0.9 mg/ml. Dithionite reduced minus oxidized spectra were determined (15Hurst J.K. Loehr T.M. Curnutte J.T. Rosen H. J. Biol. Chem. 1991; 266: 1627-1634Abstract Full Text PDF PubMed Google Scholar). Representative experiments of three determinations are shown.View Large Image Figure ViewerDownload Hi-res image Download (PPT)We considered that the failure to insert heme into FRE1 might destabilize the protein, making it difficult to distinguish mutations affecting heme liganding from mutations affecting protein stability. Therefore, we evaluated FRE1 protein expression in the histidine mutants. The mutations did not significantly affect the level of FRE1 protein as assessed by immunoblotting (Fig. 3), making it likely that abrogation of the heme spectrum was a specific consequence of the point mutation and not the result of protein destabilization.Fig. 3FRE1 protein levels expressed by the wild-type and mutant FRE1 alleles. Strain 499Δ1Δ2 was transformed with plasmids lacking FRE1, carrying the wild-type FRE1 allele, the critical histidine mutations H294A, H308A, H364A, H378A, or HPFT histidine mutant H462A. The transformants were induced for ferric reductase activity. Cell lysates (alkali lysis) were separated by SDS-PAGE, blotted onto nitrocellulose filters, and probed with a FRE1 anti-peptide antibody followed by enhanced chemiluminescence. Lanes as in Fig. 2.View Large Image Figure ViewerDownload Hi-res image Download (PPT)DISCUSSIONThe data presented here support a common model for the heme coordination in FRE1 and gp91phox proteins. Most likely there are two hemes per protein monomer. Each heme is coordinated between a pair of histidine residues located within distinct α-helical transmembrane domains separated by an intervening transmembrane domain. The 12-13 amino acids of primary sequence separating heme-liganding histidines corresponds to the separation between the two hemes. These regions appear α-helical in character, and, thus, the imidazoles separated by 12-13 amino acids are likely to face the same side of the helix. The hemes coordinated by these histidines are likely to be situated one above the other within the membrane and perpendicular to the plane of the membrane (Fig. 4). This orientation of the hemes near opposite sides of the lipid bilayer would be expected to facilitate the transmembrane electron transport that is critical for the function of these proteins. This model resembles the model described for heme coordination in a class of organelle b cytochromes (21Widger W.R. Cramer W.A. Herrman R.G. Trebst A. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 674-678Crossref PubMed Scopus (334) Google Scholar, 23Cramer W.A. Knaff D.B. Energy Transduction in Biological Membranes: A Textbook of Bioenergetics. Springer-Verlag, New York1990: 330-335Google Scholar, 24Saraste M. FEBS Lett. 1984; 166: 367-372Crossref PubMed Scopus (126) Google Scholar).Fig. 4Structural model of stacked intramembraneous histidines coordinating two hemes. The plasma membrane is portrayed as two layers of circles with tails to indicate the phospholipid bilayer. Cylinders represent three consecutive transmembrane α helices. Imidazoles of heme-coordinating histidines are numbered according to their location in FRE1, and corresponding residues in gp91phox are 101, 115, 209, and 222. The hemes, shown as squares, contain a hexacoordinate iron (black dots). A similar model has been proposed for the mitochondrial and chloroplast b cytochromes (21Widger W.R. Cramer W.A. Herrman R.G. Trebst A. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 674-678Crossref PubMed Scopus (334) Google Scholar, 23Cramer W.A. Knaff D.B. Energy Transduction in Biological Membranes: A Textbook of Bioenergetics. Springer-Verlag, New York1990: 330-335Google Scholar, 24Saraste M. FEBS Lett. 1984; 166: 367-372Crossref PubMed Scopus (126) Google Scholar).View Large Image Figure ViewerDownload Hi-res image Download (PPT)An unexpected finding was that mutation of individual critical histidine residues in FRE1 abrogated the entire heme spectrum rather than leading to a 50% reduction. This result is difficult to reconcile with the bis-heme model described above. It is possible that disruption of the imidazole ligand of one of the hemes interferes with insertion of the second heme, due to cooperativity in the heme loading process. Alternatively, effects on protein folding or membrane insertion resulting from the failure to properly insert one of the hemes might result in loss of the second heme.The similarity between the yeast FRE1 reductase and the human NADPH oxidase helps to elucidate features of both enzymes. The residues of FRE1 required for heme binding are conserved with gp91phox, suggesting that the corresponding residues coordinate heme in the NADPH oxidase. However, the corresponding mutations in gp91phox have been uninformative. Three of the four residues corresponding to the heme-liganding residues of FRE1 have been mutated in gp91phox as experiments of nature, i.e. as naturally occurring mutations in chronic granulomatous disease patients (His101→ Arg, His209→ Tyr, His222→ Arg) (7Roos D. de Boer M. Kuribayashi F. Meischl C. Weening R.S. Segal A.W. Ahlin A. Nemet K. Hossle J.P. Bernatowska-Matuszkiewicz E. Middleton-Price H. Blood. 1996; 87: 1663-1681Crossref PubMed Google Scholar). In cells taken from each of these patients, neither the heme spectrum attributable to flavocytochrome b558 nor the mutant gp91phox protein was detected (7Roos D. de Boer M. Kuribayashi F. Meischl C. Weening R.S. Segal A.W. Ahlin A. Nemet K. Hossle J.P. Bernatowska-Matuszkiewicz E. Middleton-Price H. Blood. 1996; 87: 1663-1681Crossref PubMed Google Scholar). Thus, structure-function relationships in the NADPH oxidase could not be deduced from these naturally occurring mutations.Although the FRE1 reductase functions primarily to reduce iron and the NADPH oxidase functions to reduce oxygen, the FRE1 reductase has recently been shown to be capable of inefficient oxygen reduction (9Lesuisse E. Casteras-Simon M. Labbe P. J. Biol. Chem. 1996; 271: 13578-13583Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). Introduction of components of the NADPH oxidase into yeast therefore might provide a setting for studying the requirements for efficient superoxide generation. The requirements for heme insertion into the active membrane complex could also be studied in yeast. Defining and satisfying these requirements will be important for efforts to reconstitute the NADPH oxidase in patients with chronic granulomatous disease.The shared bis-heme binding motif raises the question of the evolutionary and functional relationships between the cell surface membrane proteins gp91phox and FRE1 and the organelle b cytochromes. At the level of primary amino acid sequence, little more than the spacing of the histidines in a generally hydrophobic context is conserved between these two groups of proteins. However, a more closely related protein family can be defined, including FRE1 and gp91phox. The members of this family possess hydropathy profiles with multiple (six to eight) hydrophobic domains at the amino terminus. The bis-heme binding motif occurs in this region. A hydrophilic carboxyl-terminal domain characterizes these proteins and is absent from the organelle b cytochromes. Within this hydrophilic tail are a number of conserved amino acid motifs that may relate to cofactor binding (NADPH and FAD) (19Roman D.G. Dancis A. Anderson G.J. Klausner R.D. Mol. Cell. Biol. 1993; 13: 4342-4350Crossref PubMed Scopus (110) Google Scholar, 25Segal A.W. Abo A. Trends Biochem. Sci. 1993; 18: 43-47Abstract Full Text PDF PubMed Scopus (562) Google Scholar) and that appear in the same order and with similar spacing. This protein family includes the homologs of the gp91phox gene from various other species including pigs, mice, and even plants (26Groom Q.J. Torres M.A. Fordham-Skelton A.P. Hammond-Kosack K.E. Robinson N.J. Jones J.D.G. Plant J. 1996; 10: 515-522Crossref PubMed Scopus (243) Google Scholar), and these genes presumably function as part of a superoxide generating complex in cellular defense. A computer search of the recently completed Saccharomyces Genome Data Base for homologs of FRE1 identified FRE2 and seven additional open reading frames that appear to belong to the same gene family (Table I). These newly identified yeast genes resemble FRE1 to varying degrees but show little similarity to the organelle b cytochromes. The role of the bis-heme binding motif will be further clarified by definition of the cellular localization and function of the members of this gene family. It is highly likely that transmembrane electron transport will be involved in the function of these genes.Table ISaccharomyces cerevisiae FRE1 familyGene (identifier)FASTA score (versus FRE1)Motifs (number of initial amino acid)Bis-hemeHP(F/Y)(T/S)GP(F/Y)GCG(P/S)FRE1 YLR214w4593294462514652FRE2 YKL220c565316479526677YNR060w574309472519685YLL051c572323493538678YOR381w566316479526677YOR384w566310473520660YOL152w351197369411YGL160w254136306543YLR047c149152320CYTb PIR A001594382 Open table in a new tab INTRODUCTIONThe human phagocyte NADPH oxidase is a heme-containing enzyme complex critical for defense against microorganisms (1Thrasher A.J. Keep N.H. Wientjes F. Segal A.W. Biochim. Biophys. Acta. 1994; 1227: 1-24Crossref PubMed Scopus (212) Google Scholar). Defective functioning of this oxidase results in the susceptibility to infection that characterizes patients with chronic granulomatous disease. The genetic defects affecting the oxidase are currently the focus of corrective gene therapy trials (2Thrasher A.J. de Alwis M. Casimir C.M. Kinnon C. Page K. Lebkowski J. Segal A.W. Levinsky R.J. Blood. 1995; 86: 761-765Crossref PubMed Google Scholar, 3Li F. Linton G.F. Sekhsaria S. Whiting-Theobald N. Katkin J.P. Gallin J.I. Malech H.L. Blood. 1994; 84: 53-58Crossref PubMed Google Scholar). The NADPH oxidase assembles from two membrane (gp91phox and p22phox which together comprise flavocytochrome b558) and three cytosolic (p67phox, p47phox, and p40phox) components in response to a signaling pathway mediated through the small GTP-binding protein Rac (1Thrasher A.J. Keep N.H. Wientjes F. Segal A.W. Biochim. Biophys. Acta. 1994; 1227: 1-24Crossref PubMed Scopus (212) Google Scholar). The heme cofactor is required for enzymatic function: the transfer of electrons from cytosolic NADPH across the vacuolar membrane to molecular oxygen. The superoxide generated by this process in turn gives rise to toxic byproducts which are used in cellular defense against ingested bacteria and fungi (1Thrasher A.J. Keep N.H. Wientjes F. Segal A.W. Biochim. Biophys. Acta. 1994; 1227: 1-24Crossref PubMed Scopus (212) Google Scholar).Despite efforts by several laboratories, the sites within the oxidase where the essential heme cofactors are bound have not been defined (4Quinn M.T. Mullen J.L. Jesaitis A.J. J. Biol. Chem. 1992; 267: 7303-7309Abstract Full Text PDF PubMed Google Scholar, 5Cross A.R. Rae J. Curnutte J.T. J. Biol. Chem. 1995; 270: 17075-17077Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 6Segal A.W. Immunodeficiency. 1993; 4: 167-179PubMed Google Scholar). Heme copurifies with the cytochrome b subunits, gp91phox and p22phox (6Segal A.W. Immunodeficiency. 1993; 4: 167-179PubMed Google Scholar). However, inherited mutations in either of these subunits are associated with the absence of detectable heme in the enzyme complex and destabilization of the proteins, thus frustrating efforts to identify the specific heme-liganding residues (7Roos D. de Boer M. Kuribayashi F. Meischl C. Weening R.S. Segal A.W. Ahlin A. Nemet K. Hossle J.P. Bernatowska-Matuszkiewicz E. Middleton-Price H. Blood. 1996; 87: 1663-1681Crossref PubMed Google Scholar).We undertook to address this problem via study of the FRE1 reductase of the yeast Saccharomyces cerevisiae. FRE1 is homologous to gp91phox (8Dancis A. Roman D.G. Anderson G.J. Hinnebusch A.G. Klausner R.D. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 3869-3873Crossref PubMed Scopus (281) Google Scholar) and encodes a plasma membrane-associated subunit of an iron reductase (9Lesuisse E. Casteras-Simon M. Labbe P. J. Biol. Chem. 1996; 271: 13578-13583Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). FRE1 or FRE2 is required for iron uptake in yeast (10Georgatsou E. Alexandraki D. Mol. Cell. Biol. 1994; 14: 3065-3073Crossref PubMed Scopus (197) Google Scholar). FRE1 is required for the major (90%) extracellular ferric reductase activity under most conditions and appears to be a structural subunit of the externally directed reductase activity (8Dancis A. Roman D.G. Anderson G.J. Hinnebusch A.G. Klausner R.D. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 3869-3873Crossref PubMed Scopus (281) Google Scholar). Chelated ferric iron outside the cell is reduced by this activity to ferrous iron and then transported into the cell by the high-affinity iron uptake system (11Stearman R. Yuan D.S. Yamaguchi-Iwai Y. Klausner R.D. Dancis A. Science. 1996; 271: 1552-1557Crossref PubMed Scopus (574) Google Scholar). Thus, this iron reductase of yeast, like the NADPH oxidase of humans, is involved in the transfer of electrons from a cytosolic donor across a membrane to an extracellular acceptor.The yeast iron reductase, like the human NADPH oxidase, requires heme for its function. The requirement of heme for the plasma membrane ferric reductase of yeast is supported by the lack of activity in mutants deficient in heme biosynthesis (12Lesuisse E. Labbe P. J. Gen. Microbiol. 1989; 135: 257-263PubMed Google Scholar). In addition, FRE1 expression levels correlate with the visible spectrum attributed to the b heme cofactor (13Shatwell K.P. Dancis A. Cross A.R. Klausner R.D. Segal A.W. J. Biol. Chem. 1996; 271: 14240-14244Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). The appearance of this visible spectrum is extremely similar to the spectrum of the purified NADPH oxidase b cytochrome (13Shatwell K.P. Dancis A. Cross A.R. Klausner R.D. Segal A.W. J. Biol. Chem. 1996; 271: 14240-14244Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). Two hemes are probably present in each complex of the NADPH oxidase, as indicated by quantitation of heme in the purified cytochrome (4Quinn M.T. Mullen J.L. Jesaitis A.J. J. Biol. Chem. 1992; 267: 7303-7309Abstract Full Text PDF PubMed Google Scholar, 14Segal A.W. West I. Wientjes F. Nugent J.H.A. Chavan A.J. Haley B. Garcia R.C. Rosen H. Scrace G. Biochem. J. 1992; 284: 781-788Crossref PubMed Scopus (289) Google Scholar). Midpoint potential titration of the gp91phox Arg54→ Ser mutant is also consistent with the presence of two hemes with potentials of −220 and −300 mV (5Cross A.R. Rae J. Curnutte J.T. J. Biol. Chem. 1995; 270: 17075-17077Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar), and subsequent reanalysis of the data for the normal protein indicates the presence of two low potential hemes of −225 and −265 mV (5Cross A.R. Rae J. Curnutte J.T. J. Biol. Chem. 1995; 270: 17075-17077Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). Data from resonance Raman spectroscopy suggests a bis-imidazole coordination (15Hurst J.K. Loehr T.M. Curnutte J.T. Rosen H. J. Biol. Chem. 1991; 266: 1627-1634Abstract Full Text PDF" @default.
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• W2060193350 date "1996-12-01" @default.
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• W2060193350 title "Intramembrane Bis-Heme Motif for Transmembrane Electron Transport Conserved in a Yeast Iron Reductase and the Human NADPH Oxidase" @default.
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