David M. Dooley

A Crosslinked Cofactor in Lysyl Oxidase: Redox Function for Amino Acid Side Chains

A previously unknown redox cofactor has been identified in the active site of lysyl oxidase from the bovine aorta. Edman sequencing, mass spectrometry, ultraviolet-visible spectra, and resonance Raman studies showed that this cofactor is a quinone. Its structure is derived from the crosslinking of the ϵ-amino group of a peptidyl lysine with the modified side chain of a tyrosyl residue, and it has been designated lysine tyrosylquinone. This quinone appears to be the only example of a mammalian cofactor formed from the crosslinking of two amino acid side chains. This discovery expands the range of known quino-cofactor structures and has implications for the mechanism of their biogenesis.

Crystal structure of a eukaryotic (pea seedling) copper-containing amine oxidase at 2.2 A resolution.

BACKGROUND Copper-containing amine oxidases catalyze the oxidative deamination of primary amines to aldehydes, in a reaction that requires free radicals. These enzymes are important in many biological processes, including cell differentiation and growth, would healing, detoxification and signalling. The catalytic reaction requires a redox cofactor, topa quinone (TPQ), which is derived by post-translational modification of an invariant tyrosine residue. Both the biogenesis of the TPQ cofactor and the reaction catalyzed by the enzyme require the presence of a copper atom at the active site. The crystal structure of a prokaryotic copper amine oxidase from E. coli (ECAO) has recently been reported. RESULTS The first structure of a eukaryotic (pea seedling) amine oxidase (PSAO) has been solved and refined at 2.2 A resolution. The crystallographic phases were derived from a single phosphotungstic acid derivative. The positions of the tungsten atoms in the W12 clusters were obtained by molecular replacement using E. coli amine oxidase as a search model. The methodology avoided bias from the search model, and provides an essentially independent view of a eukaryotic amine oxidase. The PSAO molecule is a homodimer; each subunit has three domains. The active site of each subunit lies near an edge of the beta-sandwich of the largest domain, but is not accessible from the solvent. The essential active-site copper atom is coordinated by three histidine side chains and two water molecules in an approximately square-pyramidal arrangement. All the atoms of the TPQ cofactor are unambiguously defined, the shortest distance to the copper atom being approximately 6 A. CONCLUSIONS There is considerable structural homology between PSAO and ECAO. A combination of evidence from both structures indicates that the TPQ side chain is sufficiently flexible to permit the aromatic grouf to rotate about the Cbeta-Cgamma bond, and to move between bonding and non-bonding positions with respect to the Cu atom. Conformational flexibility is also required at the surface of the molecule to allow the substrates access to the active site, which is inaccessible to solvent, as expected for an enzyme that uses radical chemistry.

Copper-containing oxidases.

Major advances have been made during 1997 and 1998 toward understanding the structure/function relationships of the active sites in copper-containing oxidases. Central to this progress has been the elucidation of crystal structures for many of these enzymes. For example, studies of the mechanisms of biogenesis and/or catalysis of amine oxidase and galactose oxidase have been both stimulated and directed by the availability of structures for these proteins. Similarly, it is anticipated that the recently published crystal structures of peptidylglycine alpha-hydroxylating monooxygenase and laccase will contribute greatly toward understanding the roles of copper in these two proteins.

Pathway for Heme Uptake from Human Methemoglobin by the Iron-regulated Surface Determinants System of Staphylococcus aureus

The iron-regulated surface proteins IsdA, IsdB, and IsdC and transporter IsdDEF of Staphylococcus aureus are involved in heme acquisition. To establish an experimental model of heme acquisition by this system, we have investigated hemin transfer between the various couples of human methemoglobin (metHb), IsdA, IsdB, IsdC, and IsdE by spectroscopic and kinetic analyses. The efficiencies of hemin transfer from hemin-containing donors (holo-protein) to different hemin-free acceptors (apo-protein) were examined, and the rates of the transfer reactions were compared with that of indirect loss of hemin from the relevant donor to H64Y/V68F apomyoglobin. The efficiencies, spectral changes, and kinetics of the transfer reactions demonstrate that: 1) metHb directly transfers hemin to apo-IsdB, but not to apo-IsdA, apo-IsdC, and apo-IsdE; 2) holo-IsdB directly transfers hemin to apo-IsdA and apo-IsdC, but not to apo-IsdE; 3) apo-IsdE directly acquires hemin from holo-IsdC, but not from holo-IsdB and holo-IsdA; and 4) IsdB and IsdC enhance hemin transfer from metHb to apo-IsdC and from holo-IsdB to apo-IsdE, respectively. Taken together with our recent finding that holo-IsdA directly transfers its hemin to apo-IsdC, these results provide direct experimental evidence for a model in which IsdB acquires hemin from metHb and transfers it directly or through IsdA to IsdC. Hemin is then relayed to IsdE, the lipoprotein component of the IsdDEF transporter.

Direct Hemin Transfer from IsdA to IsdC in the Iron-regulated Surface Determinant (Isd) Heme Acquisition System of Staphylococcus aureus

The iron-regulated surface determinants (Isd) of Staphylococcus aureus, including surface proteins IsdA, IsdB, IsdC, and IsdH and ATP-binding cassette transporter IsdDEF, constitute the machinery for acquiring heme as a preferred iron source. Here we report hemin transfer from hemin-containing IsdA (holo-IsdA) to hemin-free IsdC (apo-IsdC). The reaction has an equilibrium constant of 10 ± 5 at 22 °C in favor of holo-IsdC formation. During the reaction, holo-IsdA binds to apo-IsdC and then transfers the cofactor to apo-IsdC with a rate constant of 54.3 ± 1.8 s–1 at 25 °C. The transfer rate is >70,000 times greater than the rate of simple hemin dissociation from holo-IsdA into solvent (ktransfer = 54.3 s–1 versus k–hemin = 0.00076 s–1). The standard free energy change, ΔG0, is –27 kJ/mol for the formation of the holo-IsdA-apo-IsdC complex. IsdC has a higher affinity for hemin than IsdA. These results indicate that the IsdA-to-IsdC hemin transfer is through the activated holo-IsdA-apo-IsdC complex and is driven by the higher affinity of apo-IsdC for the cofactor. These findings demonstrate for the first time in the Isd system that heme transfer is rapid, direct, and affinity-driven from IsdA to IsdC. These results also provide the first example of heme transfer from one surface protein to another surface protein in Gram-positive bacteria and, perhaps most importantly, indicate that the mechanism of activated heme transfer, which we previously demonstrated between the streptococcal proteins Shp and HtsA, may apply in general to all bacterial heme transport systems.

A model of the copper centres of nitrous oxide reductase (Pseudomonas stutzeri)

Nitrous oxide reductase (N2OR), Pseudomonas stutzeri, catalyses the 2 electron reduction of nitrous oxide to di‐nitrogen. The enzyme has 2 identical subunits (M 1 ∼ 70 000) of known amino acid sequence and contains ∼ 4 Cu ions per subunit. By measurement of the optical absorption, electron paramagnetic resonance (EPR) and low‐temperature magnetic circular dichroism (MCD) spectra of the oxidised state, a semi‐reduced form and the fully reduced state of the enzyme it is shown that the enzyme contains 2 distinct copper centres of which one is assigned to an electron‐transfer function, centre A, and the other to a catalytic site, centre Z. The latter is a binuclear copper centre with at least 1 cysteine ligand and cycles between oxidation levels Cu(II)/Cu(II) and Cu(II)/Cu(I) in the absence of substrate or inhibitors. The state Cu(II)/Cu(I) is enzymatically inactive. The MCD spectra provide evidence for a second form of centre Z, which may be enzymatically active, in the oxidised state of the enzyme. Centre A is structurally similar to that of CuA in bovine and bacterial cytochrome c oxidase and also contains copper ligated by cysteine. This centre may also be a binuclear copper complex.

The Mechanism of Direct Heme Transfer from the Streptococcal Cell Surface Protein Shp to HtsA of the HtsABC Transporter

The heme-binding proteins Shp and HtsA are part of the heme acquisition machinery found in Streptococcus pyogenes. The hexacoordinate heme (Fe(II)-protoporphyrin IX) or hemochrome form of holoShp (hemoShp) is stable in air in Tris-HCl buffer, pH 8.0, binds to apoHtsA with a Kd of 120 ± 18 μm, and transfers its heme to apoHtsA with a rate constant of 28 ± 6s–1 at 25 °C, pH 8.0. The hemoHtsA product then autoxidizes to the hexacoordinate hemin (Fe(III)-protoporphyrin IX) or hemichrome form (hemiHtsA) with an apparent rate constant of 0.017 ± 0.002 s–1. HemiShp also rapidly transfers hemin to apoHtsA through a hemiShp·apoHtsA complex (Kd = 48 ± 7 μm) at a rate ∼40,000 times greater than the rate of simple hemin dissociation from hemiShp into solvent (ktransfer = 43 ± 3s–1 versus k–hemin = 0.0003 ± 0.00006 s–1). The rate constants for hemin binding to and dissociation from HtsA (k′hemin ≈ 80 μm–1 s–1, k–hemin = 0.0026 ± 0.0002 s–1) are 50- and 10-fold greater than the corresponding rate constants for Shp (khemin ≈ 1.6 μm–1 s–1, k–hemin = 0.0003 s–1), which implies that HtsA has a more accessible active site. However, the affinity of apoHtsA for hemin (khemin ≈ 31,000 μm–1) is roughly 5-fold greater than that of apoShp (khemin ≈ 5,300 μm–1), accounting for the net transfer from Shp to HstA. These results support a direct, rapid, and affinity-driven mechanism of heme and hemin transfer from the cell surface receptor Shp to the ATP-binding cassette transporter system.

Crystal structure of the precursor of galactose oxidase: An unusual self-processing enzyme

Galactose oxidase (EC 1.1.3.9) is a monomeric enzyme that contains a single copper ion and catalyses the stereospecific oxidation of primary alcohols to their corresponding aldehydes. The protein contains an unusual covalent thioether bond between a tyrosine, which acts as a radical center during the two-electron reaction, and a cysteine. The enzyme is produced in a precursor form lacking the thioether bond and also possessing an additional 17-aa pro-sequence at the N terminus. Previous work has shown that the aerobic addition of Cu2+ to the precursor is sufficient to generate fully processed mature enzyme. The structure of the precursor protein has been determined to 1.4 Å, revealing the location of the pro-sequence and identifying structural differences between the precursor and the mature protein. Structural alignment of the precursor and mature forms of galactose oxidase shows that five regions of main chain and some key residues of the active site differ significantly between the two forms. The precursor structure provides a starting point for modeling the chemistry of thioether bond formation and pro-sequence cleavage.

Copper-tyrosyl radical enzymes.

Advances have been made since 2000 that contribute to our understanding of the biogenesis, structure and mechanism of copper-containing tyrosyl radical enzymes. Efforts to detail the biogenesis of galactose oxidase have produced the structure of the precursor enzyme, which provides a framework for emerging mechanistic studies. The role of the tyrosyl radical of cytochrome c oxidase is being defined in studies that aim to understand the His-Tyr crosslink, the location of the radical and, by direct attempts, to provide evidence for the radical during turnover.

Structure and inhibition of human diamine oxidase

Humans have three functioning genes that encode copper-containing amine oxidases. The product of the AOC1 gene is a so-called diamine oxidase (hDAO), named for its substrate preference for diamines, particularly histamine. hDAO has been cloned and expressed in insect cells and the structure of the native enzyme determined by X-ray crystallography to a resolution of 1.8 A. The homodimeric structure has the archetypal amine oxidase fold. Two active sites, one in each subunit, are characterized by the presence of a copper ion and a topaquinone residue formed by the post-translational modification of a tyrosine. Although hDAO shares 37.9% sequence identity with another human copper amine oxidase, semicarbazide sensitive amine oxidase or vascular adhesion protein-1, its substrate binding pocket and entry channel are distinctly different in accord with the different substrate specificities. The structures of two inhibitor complexes of hDAO, berenil and pentamidine, have been refined to resolutions of 2.1 and 2.2 A, respectively. They bind noncovalently in the active-site channel. The inhibitor binding suggests that an aspartic acid residue, conserved in all diamine oxidases but absent from other amine oxidases, is responsible for the diamine specificity by interacting with the second amino group of preferred diamine substrates.