The N-lobe of human serum transferrin (hTF/2N) has been expressed in baby hamster kidney cells and crystallized in both orthorhombic (P212121) and tetragonal (P41212) space groups. Both crystal forms diffract to high resolution (1.6 and 1.8 A, respectively) and have been solved by molecular replacement. Subsequent refinement resulted in final models for the structure of hTF/2N that had crystallographic R-factors of 18.1 and 19.7% for the two crystal forms, respectively; these models represent the highest-resolution transferrin structures determined to date. The hTF/2N polypeptide has a folding pattern similar to those of other transferrins, including the presence of a deep cleft that contains the metal-binding site. In contrast to other transferrins, both crystal forms of hTF/2N display disorder at the iron-binding site; model building suggests that this disorder consists of alternative conformations of the synergistically bound carbonate anion, the side chain for Arg-124, and several solvent molecules. Subsequent refinement revealed that conformation A has an occupancy of 0.63-0. 65 and corresponds to the structure of the iron-binding site found in other transferrins. The alternative conformation B has an occupancy of 0.35-0.37; in this structure, the carbonate has rotated 30 degrees relative to the iron and the side chain for Arg-124 has moved to accommodate the new carbonate position. Several water molecules appear to stabilize the carbonate anion in the two conformations. These structures are consistent with the protonation of the carbonate and resulting partial removal of the anion from the metal; these events would occur prior to cleft opening and metal release.

Ceruloplasmin is an abundant alpha 2-serum glycoprotein that contains 95% of the copper found in the plasma of vertebrate species. We report here on the identification of a genetic defect in the ceruloplasmin gene in a patient previously noted to have a total absence of circulating serum ceruloplasmin in association with late-onset retinal and basal ganglia degeneration. In this patient T2 (transverse relaxation time)-weighted magnetic resonance imaging of the brain revealed basal ganglia densities consistent with iron deposition, and liver biopsy confirmed the presence of excess iron. Although Southern blot analysis of the patient's DNA was normal, PCR amplification of 18 of the 19 exons composing the human ceruloplasmin gene revealed a distinct size difference in exon 7. DNA sequence analysis of this exon revealed a 5-bp insertion at amino acid 410, resulting in a frame-shift mutation and a truncated open reading frame. The validity of this mutation was confirmed by analysis of DNA from the patient's daughter, which revealed heterozygosity for this same 5-bp insertion. The presence of this mutation in conjunction with the clinical and pathologic findings demonstrates an essential role for ceruloplasmin in human biology and identifies aceruloplasminemia as an autosomal recessive disorder of iron metabolism. These findings support previous studies that identified ceruloplasmin as a ferroxidase and are remarkably consistent with recent studies on the essential role of a homologous copper oxidase in iron metabolism in yeast. The clinical and laboratory findings suggest that additional patients with movement disorders and nonclassical Wilson disease should be examined for ceruloplasmin gene mutations.

The DNA sequences of a complementary deoxyribonucleic acid (cDNA) and a portion of the gene coding for human prothrombin have been determined. The cDNA was 2005 base pairs in length and was found to code for part of a leader sequence of 36 amino acids, 579 amino acids present in the mature protein, a stop codon, a noncoding region of 97 base pairs, and a poly(A) tail of 27 base pairs. It is proposed that the leader sequence consists of a signal sequence and a pro sequence for the mature protein that circulates in plasma. The 10 glutamic acid residues that are present in the amino-terminal region of prothrombin and are converted to gamma-carboxyglutamic acid in the mature protein are coded by only the GAG codon. The cDNA for prothrombin was also employed as a probe for screening a human fetal liver genomic DNA library. One of the strongly positive phage containing a human DNA insert of 5 kilobases was mapped with restriction endonucleases and sequenced. This DNA contained approximately half of the gene for human prothrombin and included six introns and five exons coding for amino acid residues 144-448. The two largest intervening sequences in the genomic DNA contained two copies each of AluI repetitive DNA.

Serum transferrin binds ferric ions in the bloodstream and transports them to cells, where they are released in a process involving receptor-mediated endocytosis. Iron release is believed to be pH dependent and is coupled with a large conformational change. To help define the steps in iron release, we have determined the three-dimensional structure of the iron-free (apo) form of the recombinant N-lobe half-molecule of human serum transferrin (ApoTfN) by X-ray crystallography. Two crystal forms were obtained, form 1 with four molecules in the asymmetric unit and form 2 with two molecules in the asymmetric unit. The structures of both forms were determined by molecular replacement and were refined at 2.2 and 3.2 A resolution, respectively. Final R-factors were 0.203 (free R = 0. 292) for form 1 and 0.217 (free R = 0.312) for form 2. All six copies of the ApoTfN structure are essentially identical. Comparison with the holo form (FeTfN) shows that a large rigid-body domain movement of 63 degrees has occurred in ApoTfN, to give an open binding cleft. The extent of domain opening is the same as in the N-lobe of human lactoferrin, showing that it depends on internal constraints that are conserved in both proteins, and that it is unaffected by the presence or absence of the C-lobe. Although the conformational change is primarily a rigid-body motion, several local adjustments occur. In particular, two iron ligands, Asp 63 and His 249, change conformation to form salt bridges, with Lys 296 and Glu 83, respectively, in the binding cleft of the apo protein. Both salt bridges would have to break for iron coordination to occur. Most importantly, the structure, determined at a pH (5.3) that is close to the pH of physiological iron release, indicates that protonation of His 249 is a key step in iron release.

Transition metal ions are essential nutrients to all forms of life. Iron, copper, zinc, manganese, cobalt and nickel all have unique chemical and physical properties that make them attractive molecules for use in biological systems. Many of these same properties that allow these metals to provide essential biochemical activities and structural motifs to a multitude of proteins including enzymes and other cellular constituents also lead to a potential for cytotoxicity. Organisms have been required to evolve a number of systems for the efficient uptake, intracellular transport, protein loading and storage of metal ions to ensure that the needs of the cells can be met while minimizing the associated toxic effects. Disruptions in the cellular systems for handling transition metals are observed as a number of diseases ranging from hemochromatosis and anemias to neurodegenerative disorders including Alzheimer's and Parkinson's disease. The yeast Saccharomyces cerevisiae has proved useful as a model organism for the investigation of these processes and many of the genes and biological systems that function in yeast metal homeostasis are conserved throughout eukaryotes to humans. This review focuses on the biological roles of iron, copper, zinc, manganese, nickel and cobalt, the homeostatic mechanisms that function in S. cerevisiae and the human diseases in which these metals have been implicated.

The unique structural feature of the dilysine (Lys206-Lys296) pair in the transferrin N-lobe (hTF/2N) has been postulated to serve a special function in the release of iron from the protein. These two lysines, which are located in opposite domains, hydrogen bond to each other in the iron-containing hTF/2N at neutral pH but are far apart in the apo-form of the protein. It has been proposed that charge repulsion resulting from the protonation of the dilysines at lower pH may be the trigger to open the cleft and facilitate iron release. The fact that the dilysine pair is positively charged and resides in a location close to the metal-binding center has also led to the suggestion that the dilysine pair is an anion-binding site for chelators. The present report provides comprehensive evidence to confirm that the dilysine pair plays this dual role in modulating release of iron. When either of the lysines is mutated to glutamate or glutamine or when both are mutated to glutamate, release of iron is much slower compared to the wild-type protein. This is due to the fact that the driving force for cleft opening is absent in the mutants or is converted to a lock-like interaction (in the case of the K206E and K296E mutants). Direct titration of the apo-proteins with anions as well as anion-dependent iron release studies show that the dilysine pair is part of an active anion-binding site which exists with the Lys296-Tyr188 interaction as a core. At this site, Lys296 serves as the primary anion-binding residue and Tyr188 is the main reporter for electronic spectral change, with smaller contributions from Lys206, Tyr85, and Tyr95. In iron-loaded hTF/2N, anion binding becomes invisible as monitored by UV-vis difference spectra since the spectral reporters Tyr188 and Tyr95 are bound to iron. Our data strongly support the hypothesis that the apo-hTF/2N exists in equilibrium between the open and closed conformations, because only in the closed form is Lys296 in direct contact with Tyr188. The current findings bring together observations, ideas, and experimental data from a large number of previous studies and shed further light on the detailed mechanism of iron release from the transferrin N-lobe. In iron-containing hTF/2N, Lys296 may still function as a target to introduce an anion (or a chelator) near to the iron-binding center. When the pH is lowered, the protonation of carbonate (synergistic anion for metal binding) and then the dilysine pair form the driving force to loosen the cleft, exposing iron; the nearby anion (or chelator) then binds to the iron and releases it from the protein.

Residue Asn57 of bovine liver cytochrome b5 has been replaced with a cysteine residue, and the resulting variant has been isolated from recombinant Escherichia coli as a mixture of four major species: A, BI, BII, and C. A combination of electronic spectroscopy, 'H NMR spectroscopy, resonance Raman spectroscopy, electrospray mass spectrometry, and direct electrochemistry has been used to characterize these four major cytochrome derivatives. The red form A (Em = -19 mV) is found to possess a heme group bound covalently through a thioether linkage involving Cys57 and the a carbon of the heme 4-vinyl group. Form B, has a covalently bound heme group coupled through a thioether linkage involving the 13 carbon of the heme 4-vinyl group. Form BI, is similar to B, except that the sulfur involved in the thioether linkage is oxidized to a sulfoxide. The green form C (Em = 175 mV) possesses a noncovalently bound prosthetic group with spectroscopic properties characteristic of a chlorin. A mechanism is proposed for the generation of these derivatives, and the implications of these observations for the biosynthesis of cytochrome c and naturally occurring chlorin prosthetic groups are discussed.While iron-protoporphyrin IX is ubiquitous as a noncovalently bound prosthetic group in a wide variety of electrontransfer proteins and enzymes, other metalloporphyrins that vary in the type and degree of substitution in the macrocycle are known to be important in biological systems (1). These include hemes a, c, o, and d (2), the last of which represents the general class of metallochlorins-i.e., metalloporphyrins in which one pyrrole ring is reduced.Site-directed mutagenesis is an established method for generation of metalloprotein variants as a means of gaining mechanistic insight into electron-transfer processes (3). We now report a cytochrome b5 variant that is representative of a class of heme protein mutants in which the native prosthetic group undergoes specific chemical modifications. As these modifications are related to the classical designations ofheme proteins, we refer to this phenomenon in which transformation between heme types results from site-specific protein mutation as transmutation. Characterization of the present cytochrome b5 variant and its properties, combined with similar studies of subsequent variants based on related principles, promises to provide useful insight into the mechanisms involved in the covalent attachment of the heme prosthetic group to cytochromes c and in chlorin biosynthesis. The principles to be learned from this variant should lead to the design of metalloproteins with specifically modified prosthetic groups possessing useful enzymatic and/or electron-transfer properties.

Multi-copper oxidases (MCOs) are a small group of enzymes that oxidize their substrate with the concomitant reduction of dioxygen to two water molecules. Generally, multi-copper oxidases are promiscuous with regards to their reducing substrates and are capable of performing various functions in different species. To date, three multi-copper oxidases have been detected in humans—ceruloplasmin, hephaestin and zyklopen. Each of these enzymes has a high specificity towards iron with the resulting ferroxidase activity being associated with ferroportin, the only known iron exporter protein in humans. Ferroportin exports iron as Fe2+, but transferrin, the major iron transporter protein of blood, can bind only Fe3+ effectively. Iron oxidation in enterocytes is mediated mainly by hephaestin thus allowing dietary iron to enter the bloodstream. Zyklopen is involved in iron efflux from placental trophoblasts during iron transfer from mother to fetus. Release of iron from the liver relies on ferroportin and the ferroxidase activity of ceruloplasmin which is found in blood in a soluble form. Ceruloplasmin, hephaestin and zyklopen show distinctive expression patterns and have unique mechanisms for regulating their expression. These features of human multi-copper ferroxidases can serve as a basis for the precise control of iron efflux in different tissues. In this manuscript, we review the biochemical and biological properties of the three human MCOs and discuss their potential roles in human iron homeostasis.