Mapping the Initial Stages of a Protective Pathway that Enhances Catalytic Turnover by a Lytic Polysaccharide Monooxygenase.

Oxygenase and peroxygenase enzymes generate intermediates at their active sites which bring about the controlled functionalization of inert C-H bonds in substrates, such as in the enzymatic conversion of methane to methanol. To be viable catalysts, however, these enzymes must also prevent oxidative damage to essential active site residues, which can occur during both coupled and uncoupled turnover. Herein, we use a combination of stopped-flow spectroscopy, targeted mutagenesis, TD-DFT calculations, high-energy resolution fluorescence detection X-ray absorption spectroscopy, and electron paramagnetic resonance spectroscopy to study two transient intermediates that together form a protective pathway built into the active sites of copper-dependent lytic polysaccharide monooxygenases (LPMOs). First, a transient high-valent species is generated at the copper histidine brace active site following treatment of the LPMO with either hydrogen peroxide or peroxyacids in the absence of substrate. This intermediate, which we propose to be a CuII-(histidyl radical), then reacts with a nearby tyrosine residue in an intersystem-crossing reaction to give a ferromagnetically coupled (S = 1) CuII-tyrosyl radical pair, thereby restoring the histidine brace active site to its resting state and allowing it to re-enter the catalytic cycle through reduction. This process gives the enzyme the capacity to minimize damage to the active site histidine residues "on the fly" to increase the total turnover number prior to enzyme deactivation, highlighting how oxidative enzymes are evolved to protect themselves from deleterious side reactions during uncoupled turnover.

Mechanistic basis of substrate-O2 coupling within a chitin-active lytic polysaccharide monooxygenase: An integrated NMR/EPR study.

Lytic polysaccharide monooxygenases (LPMOs) have a unique ability to activate molecular oxygen for subsequent oxidative cleavage of glycosidic bonds. To provide insight into the mode of action of these industrially important enzymes, we have performed an integrated NMR/electron paramagnetic resonance (EPR) study into the detailed aspects of an AA10 LPMO-substrate interaction. Using NMR spectroscopy, we have elucidated the solution-phase structure of apo-BlLPMO10A from Bacillus licheniformis, along with solution-phase structural characterization of the Cu(I)-LPMO, showing that the presence of the metal has minimal effects on the overall protein structure. We have, moreover, used paramagnetic relaxation enhancement (PRE) to characterize Cu(II)-LPMO by NMR spectroscopy. In addition, a multifrequency continuous-wave (CW)-EPR and 15N-HYSCORE spectroscopy study on the uniformly isotope-labeled 63Cu(II)-bound 15N-BlLPMO10A along with its natural abundance isotopologue determined copper spin-Hamiltonian parameters for LPMOs to markedly improved accuracy. The data demonstrate that large changes in the Cu(II) spin-Hamiltonian parameters are induced upon binding of the substrate. These changes arise from a rearrangement of the copper coordination sphere from a five-coordinate distorted square pyramid to one which is four-coordinate near-square planar. There is also a small reduction in metal-ligand covalency and an attendant increase in the d(x2-y2) character/energy of the singly occupied molecular orbital (SOMO), which we propose from density functional theory (DFT) calculations predisposes the copper active site for the formation of a stable Cu-O2 intermediate. This switch in orbital character upon addition of chitin provides a basis for understanding the coupling of substrate binding with O2 activation in chitin-active AA10 LPMOs.

Mapping the protonation states of the histidine brace in an AA10 lytic polysaccharide monooxygenase using CW-EPR spectroscopy and DFT calculations.

The active site of the polysaccharide-degrading lytic polysaccharide monooxygenase (LPMO) enzyme features a single copper ion coordinated by a histidine brace. The primary coordination sphere of the copper contains several ligating atoms which are bonded to ionisable protons (e.g. OH2, NH2), the pKas of which are unknown. Using a combination of CW-EPR X-band spectroscopy over a range of pH values and DFT calculations, we show that the active site of a chitin-active AA10 LPMO can exist in three different protonation states (pKa1 = 8.7, pKa2 ∼ 11.5), representing the ionisation of the coordinating groups. The middle pH species (fully formed at pH ∼ 10.5) is proposed to be Cu(II)(His)2(OH)2 (N2O2 coordination) with a decoordinated R-NH3+ group at the amino terminus. This species also sees a rotation of the SOMO equatorial plane from the canonical histidine brace plane, whereby the nominal Cu d(x2 - y2)-orbital has rotated some 45° along the His-Cu(II)-His axis, driven by the elongation and decoordination of the amino group. The highest pH species (>12) is proposed to exist as a Cu(II)-azanide, in which the NH2 of the amino terminus has been deprotonated. The high pH means that this species is unlikely to be biologically relevant in the catalytic cycle of AA10 LPMOs.

C-type cytochrome-initiated reduction of bacterial lytic polysaccharide monooxygenases.

The release of glucose from lignocellulosic waste for subsequent fermentation into biofuels holds promise for securing humankind's future energy needs. The discovery of a set of copper-dependent enzymes known as lytic polysaccharide monooxygenases (LPMOs) has galvanised new research in this area. LPMOs act by oxidatively introducing chain breaks into cellulose and other polysaccharides, boosting the ability of cellulases to act on the substrate. Although several proteins have been implicated as electron sources in fungal LPMO biochemistry, no equivalent bacterial LPMO electron donors have been previously identified, although the proteins Cbp2D and E from Cellvibrio japonicus have been implicated as potential candidates. Here we analyse a small c-type cytochrome (CjX183) present in Cellvibrio japonicus Cbp2D, and show that it can initiate bacterial CuII/I LPMO reduction and also activate LPMO-catalyzed cellulose-degradation. In the absence of cellulose, CjX183-driven reduction of the LPMO results in less H2O2 production from O2, and correspondingly less oxidative damage to the enzyme than when ascorbate is used as the reducing agent. Significantly, using CjX183 as the activator maintained similar cellulase boosting levels relative to the use of an equivalent amount of ascorbate. Our results therefore add further evidence to the impact that the choice of electron source can have on LPMO action. Furthermore, the study of Cbp2D and other similar proteins may yet reveal new insight into the redox processes governing polysaccharide degradation in bacteria.

Laccases of prokaryotic origin: enzymes at the interface of protein science and protein technology.

The ubiquitous members of the multicopper oxidase family of enzymes oxidize a range of aromatic substrates such as polyphenols, methoxy-substituted phenols, amines and inorganic compounds, concomitantly with the reduction of molecular dioxygen to water. This family of enzymes can be broadly divided into two functional classes: metalloxidases and laccases. Several prokaryotic metalloxidases have been described in the last decade showing a robust activity towards metals, such as Cu(I), Fe(II) or Mn(II) and have been implicated in the metal metabolism of the corresponding microorganisms. Many laccases, with a superior efficiency for oxidation of organic compounds when compared with metals, have also been identified and characterized from prokaryotes, playing roles that more closely conform to those of intermediary metabolism. This review aims to present an update of current knowledge on prokaryotic multicopper oxidases, with a special emphasis on laccases, anticipating their enormous potential for industrial and environmental applications.

The role of Asp116 in the reductive cleavage of dioxygen to water in CotA laccase: assistance during the proton-transfer mechanism.

Multi-copper oxidases constitute a family of proteins that are capable of coupling the one-electron oxidation of four substrate equivalents to the four-electron reduction of dioxygen to two molecules of water. The main catalytic stages occurring during the process have already been identified, but several questions remain, including the nature of the protonation events that take place during the reductive cleavage of dioxygen to water. The presence of a structurally conserved acidic residue (Glu498 in CotA laccase from Bacillus subtilis) at the dioxygen-entrance channel has been reported to play a decisive role in the protonation mechanisms, channelling protons during the reduction process and stabilizing the site as a whole. A second acidic residue that is sequentially conserved in multi-copper oxidases and sited within the exit channel (Asp116 in CotA) has also been identified as being important in the protonation process. In this study, CotA laccase has been used as a model system to assess the role of Asp116 in the reduction process of dioxygen to water. The crystal structures of three distinct mutants, D116E, D116N and D116A, produced by site-saturation mutagenesis have been determined. In addition, theoretical calculations have provided further support for a role of this residue in the protonation events.

The removal of a disulfide bridge in CotA-laccase changes the slower motion dynamics involved in copper binding but has no effect on the thermodynamic stability.

The contribution of the disulfide bridge in CotA-laccase from Bacillus subtilis is assessed with respect to the enzyme's functional and structural properties. The removal of the disulfide bond by site-directed mutagenesis, creating the C322A mutant, does not affect the spectroscopic or catalytic properties and, surprisingly, neither the long-term nor the thermodynamic stability parameters of the enzyme. Furthermore, the crystal structure of the C322A mutant indicates that the overall structure is essentially the same as that of the wild type, with only slight alterations evident in the immediate proximity of the mutation. In the mutant enzyme, the loop containing the C322 residue becomes less ordered, suggesting perturbations to the substrate binding pocket. Despite the wild type and the C322A mutant showing similar thermodynamic stability in equilibrium, the holo or apo forms of the mutant unfold at faster rates than the wild-type enzyme. The picosecond to nanosecond time range dynamics of the mutant enzyme was not affected as shown by acrylamide collisional fluorescence quenching analysis. Interestingly, copper uptake or copper release as measured by the stopped-flow technique also occurs more rapidly in the C322A mutant than in the wild-type enzyme. Overall the structural and kinetic data presented here suggest that the disulfide bridge in CotA-laccase contributes to the conformational dynamics of the protein on the microsecond to millisecond timescale, with implications for the rates of copper incorporation into and release from the catalytic centres.

Secreted pectin monooxygenases drive plant infection by pathogenic oomycetes.

The oomycete Phytophthora infestans is a damaging crop pathogen and a model organism to study plant-pathogen interactions. We report the discovery of a family of copper-dependent lytic polysaccharide monooxygenases (LPMOs) in plant pathogenic oomycetes and its role in plant infection by P. infestans We show that LPMO-encoding genes are up-regulated early during infection and that the secreted enzymes oxidatively cleave the backbone of pectin, a charged polysaccharide in the plant cell wall. The crystal structure of the most abundant of these LPMOs sheds light on its ability to recognize and degrade pectin, and silencing the encoding gene in P. infestans inhibits infection of potato, indicating a role in host penetration. The identification of LPMOs as virulence factors in pathogenic oomycetes opens up opportunities in crop protection and food security.

Mechanisms underlying dioxygen reduction in laccases. Structural and modelling studies focusing on proton transfer.

BACKGROUND: Laccases are enzymes that couple the oxidation of substrates with the reduction of dioxygen to water. They are the simplest members of the multi-copper oxidases and contain at least two types of copper centres; a mononuclear T1 and a trinuclear that includes two T3 and one T2 copper ions. Substrate oxidation takes place at the mononuclear centre whereas reduction of oxygen to water occurs at the trinuclear centre. RESULTS: In this study, the CotA laccase from Bacillus subtilis was used as a model to understand the mechanisms taking place at the molecular level, with a focus in the trinuclear centre. The structures of the holo-protein and of the oxidised form of the apo-protein, which has previously been reconstituted in vitro with Cu(I), have been determined. The former has a dioxygen moiety between the T3 coppers, while the latter has a monoatomic oxygen, here interpreted as a hydroxyl ion. The UV/visible spectra of these two forms have been analysed in the crystals and compared with the data obtained in solution. Theoretical calculations on these and other structures of CotA were used to identify groups that may be responsible for channelling the protons that are needed for reduction of dioxygen to water. CONCLUSIONS: These results present evidence that Glu 498 is the only proton-active group in the vicinity of the trinuclear centre. This strongly suggests that this residue may be responsible for channelling the protons needed for the reduction. These results are compared with other data available for these enzymes, highlighting similarities and differences within laccases and multicopper oxidases.

Proximal mutations at the type 1 copper site of CotA laccase: spectroscopic, redox, kinetic and structural characterization of I494A and L386A mutants.

In the present study the CotA laccase from Bacillus subtilis has been mutated at two hydrophobic residues in the vicinity of the type 1 copper site. The mutation of Leu(386) to an alanine residue appears to cause only very subtle alterations in the properties of the enzyme indicating minimal changes in the structure of the copper centres. However, the replacement of Ile(494) by an alanine residue leads to significant changes in the enzyme. Thus the major visible absorption band is upshifted by 16 nm to 625 nm and exhibits an increased intensity, whereas the intensity of the shoulder at approx. 330 nm is decreased by a factor of two. Simulation of the EPR spectrum of the I494A mutant reveals differences in the type 1 as well as in the type 2 copper centre reflecting modifications of the geometry of these centres. The intensity weighted frequencies , calculated from resonance Raman spectra are 410 cm(-1) for the wild-type enzyme and 396 cm(-1) for the I494A mutant, indicating an increase of the Cu-S bond length in the type 1 copper site of the mutant. Overall the data clearly indicate that the Ile(494) mutation causes a major alteration of the structure near the type 1 copper site and this has been confirmed by X-ray crystallography. The crystal structure shows the presence of a fifth ligand, a solvent molecule, at the type 1 copper site leading to an approximate trigonal bipyramidal geometry. The redox potentials of the L386A and I494A mutants are shifted downwards by approx. 60 and 100 mV respectively. These changes correlate well with decreased catalytic efficiency of both mutants compared with the wild-type.

Human epidermal growth factor receptor (HER-1:HER-3) Fc-mediated heterodimer has broad antiproliferative activity in vitro and in human tumor xenografts.

All four members of the human epidermal growth factor (EGF) receptor (HER) family are implicated in human cancers. Although efficacious in a subset of patients, resistance to single-targeted anti-HER therapy [i.e., cetuximab (Erbitux) and trastuzumab (Herceptin)] is often associated with coexpression of other HER family members. This may be overcome by a HER ligand binding molecule that sequesters multiple EGF-like ligands, preventing ligand-dependent receptor activation. Toward this end, we have combined the HER-1/EGFR and HER-3 ligand binding domains, dimerized with fusion of an Fc fragment of human IgG1. This resulted in a mixture of HER-1/Fc homodimer (HFD100), HER-3/Fc homodimer (HFD300), and HER-1/Fc:HER-3/Fc heterodimer (RB200), also termed Hermodulins. The purified first-generation RB200 bound EGF and neuregulin 1 (NRG1)-beta1 ligands, determined by cross-linking and direct binding studies. The binding affinity for both was approximately 10 nmol/L by dissociation-enhanced lanthanide fluorescence immunoassay using europium (Eu)-labeled ligands. Competition studies with RB200 using Eu-EGF or Eu-NRG1-beta1 revealed that RB200 bound HER-1 ligands, including transforming growth factor-alpha and heparin-binding EGF, and HER-3 ligands NRG1-alpha and NRG1-beta3. RB200 inhibited EGF- and NRG1-beta1-stimulated tyrosine phosphorylation of HER family proteins, proliferation of a diverse range of tumor cells in monolayer cell growth assays, tumor cell proliferation as a single agent and in synergy with tyrosine kinase inhibitors, lysophosphatidic acid-stimulated cell proliferation, and tumor growth in two human tumor xenograft nude mouse models. Taken together, the data reveal that RB200 has the potential to sequester multiple HER ligands and interfere with signaling by HER-1, HER-2, and HER-3.

Purification, crystallization and preliminary X-ray study of the fungal laccase from Cerrena maxima.

Laccases are members of the blue multi-copper oxidase family that oxidize substrate molecules by accepting electrons at a mononuclear copper centre and transferring them to a trinuclear centre. Dioxygen binds to the trinuclear centre and, following the transfer of four electrons, is reduced to two molecules of water. Crystals of the laccase from Cerrena maxima have been obtained and X-ray data were collected to 1.9 A resolution using synchrotron radiation. A preliminary analysis shows that the enzyme has the typical laccase structure and several carbohydrate sites have been identified. The carbohydrate chains appear to be involved in stabilization of the intermolecular contacts in the crystal structure, thus promoting the formation of well ordered crystals of the enzyme. Here, the results of an X-ray crystallographic study on the laccase from the fungus Cerrena maxima are reported. Crystals that diffract well to a resolution of at least 1.9 A (R factor = 18.953%; R(free) = 23.835; r.m.s.d. bond lengths, 0.06 A; r.m.s.d. bond angles, 1.07 degrees) have been obtained despite the presence of glycan moieties. The overall spatial organization of C. maxima laccase and the structure of its copper-containing active centre have been determined by the molecular-replacement method using the laccase from Trametes versicolor (Piontek et al., 2002) as a structural template. In addition, four glycan-binding sites were identified and the 1.9 A X-ray data were used to determine the previously unknown primary structure of this protein. The identity (calculated from sequence alignment) between the C. maxima laccase and the T. versicolor laccase is about 87%. Tyr196 and Tyr372 show significant extra density at the ortho positions and this has been interpreted in terms of NO(2) substituents.

The 1.9 A crystal structure of heat-labile shrimp alkaline phosphatase.

Alkaline phosphatases are non-specific phosphomonoesterases that are distributed widely in species ranging from bacteria to man. This study has concentrated on the tissue-nonspecific alkaline phosphatase from arctic shrimps (shrimp alkaline phosphatase, SAP). Originating from a cold-active species, SAP is thermolabile and is used widely in vitro, e.g. to dephosphorylate DNA or dNTPs, since it can be inactivated by a short rise in temperature. Since alkaline phosphatases are zinc-containing enzymes, a multiwavelength anomalous dispersion (MAD) experiment was performed on the zinc K edge, which led to the determination of the structure to a resolution of 1.9 A. Anomalous data clearly showed the presence of a zinc triad in the active site, whereas alkaline phosphatases usually contain two zinc and one magnesium ion per monomer. SAP shares the core, an extended beta-sheet flanked by alpha-helices, and a metal triad with the currently known alkaline phosphatase structures (Escherichia coli structures and a human placental structure). Although SAP lacks some features specific for the mammalian enzyme, their backbones are very similar and may therefore be typical for other higher organisms. Furthermore, SAP possesses a striking feature that the other structures lack: surface potential representations show that the enzyme's net charge of -80 is distributed such that the surface is predominantly negatively charged, except for the positively charged active site. The negatively charged substrate must therefore be directed strongly towards the active site. It is generally accepted that optimization of the electrostatics is one of the characteristics related to cold-adaptation. SAP demonstrates this principle very clearly.

Mammary analog secretory carcinoma of salivary gland with high-grade histology arising in hard palate, report of a case and review of literature.

Mammary gland analog secretary carcinoma (MASC) of salivary gland is typically a tumor of low histologic grade and behaves as a low-grade malignancy with relatively benign course. This tumor shares histologic features, immunohistochemical profile, and a highly specific genetic translocation, ETV6-NTRK3, with secretory carcinoma of breast. Histologically, it is often mistaken as acinic cell carcinoma, adenocarcinoma not otherwise specified, and other primary salivary gland tumors. Here we report a case of MASC with high-grade transformation and cervical lymph node metastases confirmed with ETV6-NTRK3 translocation arising in the hard palate of a 41 year-old adult. Interestingly, the metastatic carcinoma has lower grade than the original tumor which strongly support malignant transformation of the original tumor. Most commonly, MASC arises from the parotid gland and less often in minor salivary glands. Metastasis is relatively uncommon and high-grade histology has only been reported in four cases with three of them arising from the parotid gland and the location of the fourth one has not been reported. This is the first case with high grade histology that arise from minor salivary gland and it emphasizes the importance of molecular screening of salivary gland tumor with high-grade histology for ETV6-NTRK3 translocation. In our literature of 115 cases that includes the current case, MASC occurred predominantly in adult with only a few cases under 18 years of age and a male to female ratio of 1.2:1. Parotid gland is more commonly affected but there is also significant occurrence in minor salivary glands. Except for the cases with high grade histology, the overall prognosis is good.

Crystal structure of the multicopper oxidase from the pathogenic bacterium Campylobacter jejuni CGUG11284: characterization of a metallo-oxidase.

Multicopper oxidases are a multi-domain family of enzymes that are able to couple oxidation of substrates with reduction of dioxygen to water. These enzymes are capable of oxidizing a vast range of substrates, varying from aromatic to inorganic compounds such as metals. This metallo-oxidase activity observed in several members of this family has been linked to mechanisms of homeostasis in different organisms. Recently, a periplasmic multicopper oxidase, encoded by Campylobacter jejuni, has been characterised and associated with copper homeostasis and with the protection against oxidative stress as it may scavenge metallic ions into their less toxic form and also inhibit the formation of radical oxygen species. In order to contribute to the understanding of its functional role, the crystal structure of the recombinant McoC (Campylobacter jejuni CGUG11284) has been determined at 1.95 Å resolution and its structural and biochemical characterizations undertaken. The results obtained indicate that McoC has the characteristic fold of a laccase having, besides the catalytic centres, another putative binding site for metals. Indeed, its biochemical and enzymatic characterization shows that McoC is essentially a metallo-oxidase, showing low enzymatic efficiency towards phenolic substrates.

New evidence for the role of calcium in the glycosidase reaction of GH43 arabinanases.

Endo-1,5-α-L-arabinanases are glycosyl hydrolases that are able to cleave the glycosidic bonds of α-1,5-L-arabinan, releasing arabino-oligosaccharides and L-arabinose. Two extracellular endo-1,5-α-L-arabinanases have been isolated from Bacillus subtilis, BsArb43A and BsArb43B (formally named AbnA and Abn2, respectively). BsArb43B shows low sequence identity with previously characterized 1,5-α-L-arabinanases and is a much larger enzyme. Here we describe the 3D structure of native BsArb43B, biochemical and structure characterization of two BsArb43B mutant proteins (H318A and D171A), and the 3D structure of the BsArb43B D171A mutant enzyme in complex with arabinohexose. The 3D structure of BsArb43B is different from that of other structurally characterized endo-1,5-α-L-arabinanases, as it comprises two domains, an N-terminal catalytic domain, with a 3D fold similar to that observed for other endo-1,5-α-L-arabinanases, and an additional C-terminal domain. Moreover, this work also provides experimental evidence for the presence of a cluster containing a calcium ion in the catalytic domain, and the importance of this calcium ion in the enzymatic mechanism of BsArb43B.

The role of Glu498 in the dioxygen reactivity of CotA-laccase from Bacillus subtilis.

The multicopper oxidases couple the one-electron oxidation of four substrate molecules to the four electron reductive cleavage of the O-O bond of dioxygen. This reduction takes place at the trinuclear copper centre of the enzyme and the dioxygen approaches this centre through an entrance channel. In this channel, an acidic residue plays a key role in steering the dioxygen to the trinuclear copper site, providing protons for the catalytic reaction and giving overall stability to this site. In this study, the role of the Glu(498) residue, located within the entrance channel to the trinuclear copper centre, has been investigated in the binding and reduction of dioxygen by the CotA-laccase from Bacillus subtilis. The absence of an acidic group at the 498 residue, as in the E498T and E498L mutants, results in a severe catalytic impairment, higher than 99%, for the phenolic and non-phenolic substrates tested. The replacement of this glutamate by aspartate leads to an activity that is around 10% relative to that of the wild-type. Furthermore, while this latter mutant shows a similar K(m) value for dioxygen, the E498T and E498L mutants show a decreased affinity, when compared to the wild-type. X-ray structural and spectroscopic analysis (UV-visible, electron paramagnetic resonance and resonance Raman) reveal perturbations of the structural properties of the catalytic centres in the Glu(498) mutants when compared to the wild-type protein. Overall, the results strongly suggest that Glu(498) plays a key role in the protonation events that occur at the trinuclear centre and in its stabilization, controlling therefore the binding of dioxygen and its further reduction.

Structural and functional relationships in the hybrid cluster protein family: structure of the anaerobically purified hybrid cluster protein from Desulfovibrio vulgaris at 1.35 A resolution.

The hybrid cluster protein (HCP) from the sulfate-reducing bacterium Desulfovibrio vulgaris strain Hildenborough has been isolated and crystallized anaerobically. The phase problem was solved for a P2(1)2(1)2(1) crystal form using multiple-wavelength anomalous diffraction data collected in the vicinity of the Fe K absorption edge. Although the overall protein structure is essentially the same as that previously obtained, it shows that the nature of the hybrid cluster has particular differences when isolated and crystallized in the absence of oxygen and this provides insight into the structural features associated with changes in the oxidation state. A comparison between HCPs and carbon monoxide dehydrogenases (CoDs) shows that they possess a similar fold and that the dehydrogenases have a related cluster at the equivalent HCP hybrid cluster position. This helps to understand the nature of the hybrid cluster and to predict a dimeric structure for class 3 HCPs, which lack the N-terminal region.

Ceruloplasmin revisited: structural and functional roles of various metal cation-binding sites.

The three-dimensional molecular structure of human serum ceruloplasmin has been reinvestigated using X-ray synchrotron data collected at 100 K from a crystal frozen to liquid-nitrogen temperature. The resulting model, with an increase in resolution from 3.1 to 2.8 A, gives an overall improvement of the molecular structure, in particular the side chains. In addition, it enables the clear definition of previously unidentified Ca2+-binding and Na+-binding sites. The Ca2+ cation is located in domain 1 in a configuration very similar to that found in the activated bovine factor Va. The Na+ sites appear to play a structural role in providing rigidity to the three protuberances on the top surface of the molecule. These features probably help to steer substrates towards the mononuclear copper sites prior to their oxidation and to restrict the size of the approaching substrate. The trinuclear copper centre appears to differ from the room-temperature structure in that a dioxygen moiety is bound in a similar way to that found in the endospore coat protein CotA from Bacillus subtilis.

Crystallographic evidence for dioxygen interactions with iron proteins.

The interaction of dioxygen with iron plays a key role in many important biological processes, such as dioxygen transport in the bloodstream and the reduction of dioxygen by iron in respiration. However, the catalytic mechanisms employed, for example in ligand oxidation, are not fully understood at the current time despite intensive biochemical, spectroscopic and structural studies. This review outlines the structural evidence obtained by X-ray crystallographic methods for the nature of the interactions between dioxygen and the metal in iron-containing proteins. Proteins involved in iron transport or electron transfer are not included.