Bovine Enzyme Research Articles

EPR and UV‐Vis studies of the Fenton reaction with the feasibility of using H2O2 generated by Abeta42 and its interaction on AChE catality activity

Two of the most important biomolecules involved with memory and learning are the acetylcholinesterase (AChE) and beta amyloid. The first one hydrolyzes the neurotransmitter acetylcholine (ACh). The second biomolecule is located in the brain forming clusters with glial cells, iron and amyloid fibrils of 42 residues (Abeta42). The production of hydrogen peroxide (H2O2) from Abeta42 fibrils, as well as the possibility that the H2O2 produced in this way it reacts with iron (Fe) via Fenton reaction, producing free radicals hydroxyl (.OH) has been proposed. These reactive oxygen species could have some effect on the AChE activity. In this work enzyme kinetics was studied by UV‐Vis, while free radical generation .OH was followed by electron paramagnetic resonance (EPR). The results show no change in the bovine AChE enzyme activity and that Abeta42 does not produce H2O2.

Nanotechnology‐Enabled Closed Loop Insulin Delivery Device: In Vitro and In Vivo Evaluation of Glucose‐Regulated Insulin Release for Diabetes Control

AbstractRecently, a new multifunctional, bio‐inorganic nanocomposite membrane with the ability to self‐regulate the release of insulin in response to blood glucose (BG) levels was reported. Herein, the application of this material as part of a small, implantable, closed‐loop insulin delivery device designed to continuously monitor BG concentrations and regulate insulin release is proposed. The insulin delivery device consists of a nanocomposite glucose‐responsive plug covalently bound to an insulin reservoir made of surface‐modified silicone. The plug is prepared with crosslinked bovine serum albumin (BSA) and enzymes (glucose oxidase (GOx) and catalase (CAT)), pH‐responsive hydrogel nanoparticles, and multifunctional MnO2 nanoparticles. The plug functions both as a glucose sensor and controlled delivery unit to release higher rates of insulin from the reservoir in response to hyperglycemic BG levels and basal insulin rates at normal BG concentration. The surfaces of the device are modified by silanization followed by PEGylation to ensure its safety and biocompatibility and the stability of encased insulin. Our results show that insulin release can be modulated in vitro in response to glucose concentrations. In vivo experiments show that the glycemia of diabetic rats can be controlled with implantation of the prototype device. The glucose‐responsiveness of the device is also demonstrated by rapid drop in BG level after challenging diabetic rats with bolus injection of glucose solution. In addition, it is demonstrated that surface PEGylation of the device is necessary for reducing the immune response of the host to the implanted foreign object and maintaining insulin stability and bioactivity. With this molecular architecture and the bio‐inorganic nanocomposite plug, the device has the ability to maintain normal BG levels in diabetic rats.

CO impedes superfast O 2 binding in ba 3 cytochrome oxidase from Thermus thermophilus

Kinetic studies of heme-copper terminal oxidases using the CO flow-flash method are potentially compromised by the fate of the photodissociated CO. In this time-resolved optical absorption study, we compared the kinetics of dioxygen reduction by ba(3) cytochrome c oxidase from Thermus thermophilus in the absence and presence of CO using a photolabile O(2)-carrier. A novel double-laser excitation is introduced in which dioxygen is generated by photolyzing the O(2)-carrier with a 355 nm laser pulse and the fully reduced CO-bound ba(3) simultaneously with a second 532-nm laser pulse. A kinetic analysis reveals a sequential mechanism in which O(2) binding to heme a(3) at 90 μM O(2) occurs with lifetimes of 9.3 and 110 μs in the absence and presence of CO, respectively, followed by a faster cleavage of the dioxygen bond (4.8 μs), which generates the P intermediate with the concomitant oxidation of heme b. The second-order rate constant of 1 × 10(9) M(-1) s(-1) for O(2) binding to ba(3) in the absence of CO is 10 times greater than observed in the presence of CO as well as for the bovine heart enzyme. The O(2) bond cleavage in ba(3) of 4.8 μs is also approximately 10 times faster than in the bovine enzyme. These results suggest important structural differences between the accessibility of O(2) to the active site in ba(3) and the bovine enzyme, and they demonstrate that the photodissociated CO impedes access of dioxygen to the heme a(3) site in ba(3), making the CO flow-flash method inapplicable.

Crystal Structures of Urate Bound Form of Xanthine Oxidoreductase: Substrate Orientation and Structure of the Key Reaction Intermediate

Two contradictory models have been proposed for the binding mode of the substrate xanthine to and its activation mechanism by xanthine oxidoreductase. In an effort to distinguish between the two models, we determined the crystal structures of the urate complexes of the demolybdo-form of the D428A mutant of rat xanthine oxidoreductase at 1.7 Å and of the reduced bovine milk enzyme at 2.1 Å, the latter representing a reaction intermediate. The results clearly indicate the catalytically relevant binding mode of the substrate xanthine.

Structure of Dimeric F1F0-ATP Synthase

The structure of the dimeric ATP synthase from yeast mitochondria was analyzed by transmission electron microscopy and single particle image analysis. In addition to the previously reported side views of the dimer, top view and intermediate projections served to resolve the arrangement of the rotary c(10) ring and the other stator subunits at the F(0)-F(0) dimeric interface. A three-dimensional reconstruction of the complex was calculated from a data set of 9960 molecular images at a resolution of 27 Å. The structural model of the dimeric ATP synthase shows the two monomers arranged at an angle of ∼45°, consistent with our earlier analysis of the ATP synthase from bovine heart mitochondria (Minauro-Sanmiguel, F., Wilkens, S., and Garcia, J. J. (2005) Proc. Natl. Acad. Sci. U.S.A. 102, 12356-12358). In the ATP synthase dimer, the two peripheral stalks are located near the F(1)-F(1) interface but are turned away from each other so that they are not in contact. Based on the three-dimensional reconstruction, a model of how dimeric ATP synthase assembles to form the higher order oligomeric structures that are required for mitochondrial cristae biogenesis is discussed.

Crystal Structures of Mutant Forms of the Yeast F1 ATPase Reveal Two Modes of Uncoupling

The mitochondrial ATP synthase couples the flow of protons with the phosphorylation of ADP. A class of mutations, the mitochondrial genome integrity (mgi) mutations, has been shown to uncouple this process in the yeast mitochondrial ATP synthase. Four mutant forms of the yeast F(1) ATPase with mgi mutations were crystallized; the structures were solved and analyzed. The analysis identifies two mechanisms of structural uncoupling: one in which the empty catalytic site is altered and in doing so, apparently disrupts substrate (phosphate) binding, and a second where the steric hindrance predicted between γLeu83 and β(DP) residues, Leu-391 and Glu-395, located in Catch 2 region, is reduced allowing rotation of the γ-subunit with less impedance. Overall, the structures provide key insights into the critical interactions in the yeast ATP synthase involved in the coupling process.

The Subunit Composition of Mitochondrial NADH:Ubiquinone Oxidoreductase (Complex I) From Pichia pastoris

Respiratory complex I (NADH:quinone oxidoreductase) is an entry point to the electron transport chain in the mitochondria of many eukaryotes. It is a large, multisubunit enzyme with a hydrophilic domain in the matrix and a hydrophobic domain in the mitochondrial inner membrane. Here we present a comprehensive analysis of the protein composition and post-translational modifications of complex I from Pichia pastoris, using a combination of proteomic and bioinformatic approaches. Forty-one subunits were identified in P. pastoris complex I, comprising the 14 core (conserved) subunits and 27 supernumerary subunits; seven of the core subunits are mitochondrial encoded. Three of the supernumerary subunits (named NUSM, NUTM, and NUUM) have not been observed previously in any species of complex I. However, homologues to all three of them are present in either Yarrowia lipolytica or Pichia angusta complex I. P. pastoris complex I has 39 subunits in common with Y. lipolytica complex I, 37 in common with N. crassa complex I, and 35 in common with the bovine enzyme. The mitochondrial encoded subunits (translated by the mold mitochondrial genetic code) retain their N-α-formyl methionine residues. At least eight subunits are N-α-acetylated, but the N-terminal modifications of the nuclear encoded subunits are not well-conserved. A combination of two methods of protein separation (SDS-PAGE and HPLC) and three different mass spectrometry techniques (peptide mass fingerprinting, tandem MS and molecular mass measurements) were required to define the protein complement of P. pastoris complex I. This requirement highlights the need for inclusive and comprehensive strategies for the characterization of challenging membrane-bound protein complexes containing both hydrophilic and hydrophobic components.

Substrate Orientation and Catalytic Specificity in the Action of Xanthine Oxidase: THE SEQUENTIAL HYDROXYLATION OF HYPOXANTHINE TO URIC ACID

Xanthine oxidase is a molybdenum-containing enzyme catalyzing the hydroxylation of a sp(2)-hybridized carbon in a broad range of aromatic heterocycles and aldehydes. Crystal structures of the bovine enzyme in complex with the physiological substrate hypoxanthine at 1.8 A resolution and the chemotherapeutic agent 6-mercaptopurine at 2.6 A resolution have been determined, showing in each case two alternate orientations of substrate in the two active sites of the crystallographic asymmetric unit. One orientation is such that it is expected to yield hydroxylation at C-2 of substrate, yielding xanthine. The other suggests hydroxylation at C-8 to give 6,8-dihydroxypurine, a putative product not previously thought to be generated by the enzyme. Kinetic experiments demonstrate that >98% of hypoxanthine is hydroxylated at C-2 rather than C-8, indicating that the second crystallographically observed orientation is significantly less catalytically effective than the former. Theoretical calculations suggest that enzyme selectivity for the C-2 over C-8 of hypoxanthine is largely due to differences in the intrinsic reactivity of the two sites. For the orientation of hypoxanthine with C-2 proximal to the molybdenum center, the disposition of substrate in the active site is such that Arg(880) and Glu(802), previous shown to be catalytically important for the conversion of xanthine to uric acid, play similar roles in hydroxylation at C-2 as at C-8. Contrary to the literature, we find that 6,8-dihydroxypurine is effectively converted to uric acid by xanthine oxidase.

The reaction of NADPH with bovine mitochondrial NADH:ubiquinone oxidoreductase revisited

Bovine NADH:ubiquinone oxidoreductase (Complex I) is the first complex in the mitochondrial respiratory chain. It has long been assumed that it contained only one FMN group. However, as demonstrated in 2003, the intact enzyme contains two FMN groups. The second FMN was proposed to be located in a conserved flavodoxin fold predicted to be present in the PSST subunit. The long-known reaction of Complex I with NADPH differs in many aspects from that with NADH. It was proposed that the second flavin group was specifically involved in the reaction with NADPH. The X-ray structure of the hydrophilic domain of Complex I from Thermus thermophilus (Sazanov and Hinchliffe 2006, Science 311, 1430-1436) disclosed the positions of all redox groups of that enzyme and of the subunits holding them. The PSST subunit indeed contains the predicted flavodoxin fold although it did not contain FMN. Inspired by this structure, the present paper describes a re-evaluation of the enigmatic reactions of the bovine enzyme with NADPH. Published data, as well as new freeze-quench kinetic data presented here, are incompatible with the general opinion that NADPH and NADH react at the same site. Instead, it is proposed that these pyridine nucleotides react at opposite ends of the 90 A long chain of prosthetic groups in Complex I. Ubiquinone is proposed to react with the Fe-S clusters in the TYKY subunit deep inside the hydrophilic domain. A new model for electron transfer in Complex I is proposed. In the accompanying paper this model is compared with the one advocated in current literature.

Ligand Trapping by Cytochrome c Oxidase: IMPLICATIONS FOR GATING AT THE CATALYTIC CENTER

Cytochrome c oxidase is a member of the heme-copper family of oxygen reductases in which electron transfer is linked to the pumping of protons across the membrane. Neither the redox center(s) associated with proton pumping nor the pumping mechanism presumably common to all heme-copper oxidases has been established. A possible conformational coupling between the catalytic center (Fe(a3)(3+)-Cu(B)(2+)) and a protein site has been identified earlier from ligand binding studies, whereas a structural change initiated by azide binding to the protein has been proposed to facilitate the access of cyanide to the catalytic center of the oxidized bovine enzyme. Here we show that cytochrome oxidase pretreated with a low concentration of azide exhibits a significant increase in the apparent rate of cyanide binding relative to that of free enzyme. However, this increase in rate does not reflect a conformational change enhancing the rapid formation of a Fe(a3)(3+)-CN-Cu(B)(2+) complex. Instead the cyanide-induced transition of a preformed Fe(a3)(3+)-N(3)-Cu(B)(2+) to the ternary complex of Fe(a3)(3+)-N(3) Cu(B)(2+)-CN is the most likely reason for the observed acceleration. Significantly, the slow rate of azide release from the ternary complex indicates that cyanide ligated to Cu(B) blocks a channel between the catalytic site and the solvent. The results suggest that there is a pathway that originates at Cu(B) and that, during catalysis, ligands present at this copper center control access to the iron of heme a(3) from the bulk medium.

Effect of dietary fatty acids on expression of lipogenic enzymes and fatty acid profile in tissues of bulls

This study investigated the effects of dietary linolenic acid (C18:3n-3) v. linoleic acid (C18:2n-6) on fatty acid composition and protein expression of key lipogenic enzymes, acetyl-CoA carboxylase (ACC), stearoyl-CoA desaturase (SCD) and delta 6 desaturase (Δ6d) in longissimus muscle and subcutaneous adipose tissue of bulls. Supplementation of the diet with C18:3n-3 was accompanied by an increased level of n-3 fatty acids in muscle which resulted in decrease of n-6/n-3 ratio. The diet enriched with n-3 polyunsaturated fatty acids (PUFAs) significantly inhibited SCD protein expression in muscle and subcutaneous adipose tissue, and reduced the Δ6d expression in muscle. There was no significant effect of the diet on ACC protein expression. Inhibition of the Δ6d expression was associated with a decrease in n-6 PUFA level in muscles, whereas repression of SCD protein was related to a lower oleic acid (C18:1 cis-9) content in the adipose tissue. Expression of ACC, SCD and Δ6d proteins was found to be relatively higher in subcutaneous adipose tissue when compared with longissimus muscle. It is suggested that dietary manipulation of fatty acid composition in ruminants is mediated, at least partially, through the regulation of lipogenic enzymes expression and that regulation of the bovine lipogenic enzymes expression is tissue specific.

Molecular Modelling Studies of Bovine and Camel Chymosin-κ-Casein Complexes

We present computational studies of two homologous mammalian aspartic proteases (calf and camel chymosin) complexed with 16-residue fragments of their native peptide ligands (cow and camel κ-casein) and the cross-complexes. Using molecular docking calculations, homology modelling and molecular dynamics simulations, we compare the binding modes of the four systems. The complexes are of industrial interest because camel chymosin has recently been marketed as an alternative to bovine chymosin as an enzyme to clot milk in cheese manufacturing. The camel enzyme has been shown to have 70% higher clotting activity and only 20% of the unspecific protease activity for bovine κ-casein as compared to the bovine enzyme. Interestingly, bovine chymosin has a very low proteolytic rate for camel κ-casein. The models provide putative atomic coordinates for these complexes, for which there are no available crystallographic or NMR structures, and help to explain some existing experimental results.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Dipeptidyl peptidase 9 (DPP9) from bovine testes: Identification and characterization as the short form by mass spectrometry

The dipeptidyl peptidases (DPP) 8 and 9 belong to the DPP4 activity and/or structure homologues (DASH). Recently, a DPP9-like protein was purified from bovine testes. The aim of the present study was to prove its identity and to investigate the characteristics of this natural enzyme. We report the identification and N-terminal sequence analysis by MALDI-TOF/TOF MS, of the purified bovine enzyme as DPP9. The tryptic peptides after in-gel digestion covered 41% and 38% of the short and full-length variants of bovine DPP9, respectively. Using Asp-N digestion combined with a very recently described mass spectrometric method using DITC glass beads, the N-terminal peptide (XTGALTSERG) was isolated. It corresponds to the N-terminus of the short form of bovine DPP9. There was no evidence for glycosylation of purified bovine DPP9. The purified DPP9 was activated and stabilized by DTT. Bovine DPP9 lost its activity almost completely after alkylation with N-ethylmaleimide. Also alkylation with iodoacetamide inhibited DPP9, albeit only 70%. Other properties of bovine DPP9 are reported, including functional stability and sensitivity towards metal ions. Our results indicate that the short form of DPP9 can be isolated from bovine testes and that it behaves as a stable enzyme suitable for further functional and biochemical characterization as well as for inhibitor screening and characterization.

Immunohistochemistry of the monkey retina with a monoclonal antibody against subunit V of cytochrome c oxidase.

A monoclonal antibody against subunit V of cytochrome c oxidase was newly prepared from purified bovine heart muscle enzyme. This antibody showed cross-reactivity against the bovine and the human cytochrome c oxidase on Western blot. Cytochrome c oxidase in paraffin embedded sections of monkey retinas and optic nerves was examined immunohistochemically using the monoclonal antibody. The inner segment of cone and rod photoreceptors, the inner nuclear layer and the inner plexiform layer showed marked reactivities. The optic nerve fiber axons also showed high reactivities, with more in the unmyelinated fibers peripheral to the lamina cribrosa. These immunohistochemical results using the antibody are mostly agreeable with previously reported histochemical localization of cytochrome c oxidase activity in the retina, but there were some noticeable differences in the plexiform layers.

Crystal structure of bovine 3‐hydroxyanthranilate 3,4‐dioxygenase

3-Hydroxyanthranilate 3,4-dioxygenase, the enzyme that catalyzes the conversion of 3-hydroxyanthranilate to quinolinic acid, has been extracted and purified from bovine kidney, crystallized and its structure determined at 2.5 A resolution. The enzyme, which crystallizes in the triclinic P1 space group, is a monomer, characterized by the so-called cupin fold. The monomer of the bovine enzyme mimics the dimer present in lower species, such as bacteria and yeast, since it is composed of two domains: one of them is equivalent to one monomer, whilst the second domain corresponds to only a portion of it. The active site consists of an iron ion coordinated by two histidine residues, one glutamate and an external ligand, which has been interpreted as a solvent molecule. It is contained in the N-terminal domain, whilst the function of the C-terminal domain is possibly structural. The catalytic mechanism very likely has been conserved through all species, since the positions of all residues considered relevant for the reaction are present from bacteria to humans.

Human recombinant hyaluronidase (Cumulase®) improves intracytoplasmic sperm injection survival and fertilization rates

The cumulus-corona-oocyte complex, composed of cumulus granulosa cells embedded in a matrix of hyaluronan oligosaccharide chains cross-linked by hyaluronan binding proteins and proteoglycans, surrounds each oocyte and must be removed prior to intracytoplasmic sperm injection (ICSI). This is traditionally achieved using enzymatic digestion of the matrix with a bovine-derived hyaluronidase followed by mechanical denudation through pipetting. A human recombinant hyaluronidase (Cumulase) has been developed with the intent of circumventing the problems and concerns associated with the animal origin and lack of purity of the bovine-derived form of the enzyme. In order to compare the effect of Cumulase with that of the bovine enzyme on the rates of normal fertilization and oocyte damage, a retrospective study using four experienced practitioners was performed. In 2006, using Cumulase, a significantly increased rate of normal fertilization (P = 0.0003) and a significantly decreased rate of oocyte damage (P < 0.0001) were observed compared with 2005, during which time bovine-derived hyaluronidase was predominantly used. This study indicates that Cumulase is safe and effective for use in the removal of the cumulus-corona-oocyte complex prior to ICSI, and may have several distinct advantages over the animal-derived form of the enzyme in terms of safety and efficacy.

New reactive polymer for protein immobilisation on sensor surfaces

Immobilisation of biorecognition elements on transducer surfaces is a key step in the development of biosensors. The immobilisation needs to be fast, cheap and most importantly should not affect the biorecognition activity of the immobilised receptor. A novel protocol for the covalent immobilisation of biomolecules containing primary amines using an inexpensive and simple polymer is presented. This tri-dimensional (3D) network leads to a random immobilisation of antibodies on the polymer and ensures the availability of a high percentage of antibody binding sites. The reactivity of the polymer is based on the reaction between primary amines and thioacetal groups included in the polymer network. These functional groups (thioacetal) do not need any further activation in order to react with proteins, making it attractive for sensor fabrication. The novel polymer also contains thiol derivative groups (disulphide groups or thioethers) that promote self-assembling on a metal transducer surface. For demonstration purposes the polymer was immobilised on Au Biacore chips. The resulting polymer layer was characterised using contact angle meter, atomic force microscopy (AFM) and ellipsometry. A general protocol suitable for the immobilisation of bovine serum albumin (BSA), enzymes and antibodies such as polyclonal anti-microcystin-LR antibody and monoclonal anti-prostate specific antigen (anti-PSA) antibody was then optimised. The affinity characteristics of developed immunosensors were investigated in reaction with microcystin-LR, and PSA. The calculated detection limit for analytes depended on the properties of antibodies. The detection limit for microcystin-LR was 10 ng mL −1 and for PSA 0.01 ng mL −1. The non-specific binding of analytes to synthesised polymers was very low. The polymer-coated chips were stored for up to 2 months without any noticeable deterioration in their ability to react with proteins. These findings make this new polymer very promising for the development of low-cost, easy to prepare and sensitive biosensors.

Cryo-EM Structure of the Yeast ATP Synthase

We have used electron cryomicroscopy of single particles to determine the structure of the ATP synthase from Saccharomyces cerevisiae. The resulting map at 24 Å resolution can accommodate atomic models of the F 1–c 10 subcomplex, the peripheral stalk subcomplex, and the N-terminal domain of the oligomycin sensitivity conferral protein. The map is similar to an earlier electron cryomicroscopy structure of bovine mitochondrial ATP synthase but with important differences. It resolves the internal structure of the membrane region of the complex, especially the membrane embedded subunits b, c, and a. Comparison of the yeast ATP synthase map, which lacks density from the dimer-specific subunits e and g, with a map of the bovine enzyme that included e and g indicates where these subunits are located in the intact complex. This new map has allowed construction of a model of subunit arrangement in the F O motor of ATP synthase that dictates how dimerization of the complex via subunits e and g might occur.

Characterization of the bovine angiotensin converting enzyme promoter: Essential roles of Egr-1, ATF-2 and Ets-1 in the regulation by phorbol ester

The protease angiotensin converting enzyme (ACE) is a key regulator of blood pressure homeostasis, and is responsible for proteolytic activation of angiotensin I to angiotensin II (Ang II), a potent vasoconstrictor, and proteolytic inactivation of bradykinin, a vasodilator. Recent studies have also implicated ACE and Ang II dysregulation in the progression of fibrotic tissue diseases. Although many studies have utilized bovine tissues and cells for investigating the regulation of ACE gene expression, the bovine ACE promoter has not been previously characterized. Here we present the analysis of the bovine ACE promoter. We investigated cis elements regulated by phorbol 12-myristate 13-acetate (PMA). Sequence analysis shows that the bovine ACE promoter contains several putative binding sites for the transcription factors ATF-2, Ets-1, Egr-1 and SP1/SP3. Chromatin immunoprecipitation (ChIP) indicated that the activation of the bovine ACE promoter by PMA involves histone H4 acetylation, and that PMA induced Egr-1 and ATF-2 binding to the ACE promoter, whereas Ets-1 binding was suppressed by PMA. The regulatory roles of these transcription factors in the bovine ACE gene regulation were confirmed by co-expression of either wild type or dominant negative transcription factors with the luciferase reporter constructs. The bovine and human ACE promoters share similarities in binding sites for transcription factors and PMA regulation within the core regions but contain significant differences in the distal promoter regions.

The Mechanism of Assembly and Cofactor Insertion into Rhodobacter capsulatus Xanthine Dehydrogenase

Rhodobacter capsulatus xanthine dehydrogenase (XDH) is a molybdo-flavoprotein that is highly homologous to the homodimeric mammalian xanthine oxidoreductase. However, the bacterial enzyme has an (alphabeta)(2) heterotetrameric structure, and the cofactors were identified to be located on two different polypeptides. We have analyzed the mechanism of cofactor insertion and subunit assembly of R. capsulatus XDH, using engineered subunits with appropriate substitutions in the interfaces. In an (alphabeta) heterodimeric XDH containing the XdhA and XdhB subunits, the molybdenum cofactor (Moco) was shown to be absent, indicating that dimerization of the (alphabeta) subunits has to precede Moco insertion. In an (alphabeta)(2) XDH heterotetramer variant, including only one active Moco-center, the active (alphabeta) site of the chimeric enzyme was shown to be fully active, revealing that the two subunits act independent without cooperativity. Amino acid substitutions at two cysteine residues coordinating FeSI of the two [2Fe-2S] clusters of the enzyme demonstrate that an incomplete assembly of FeSI impairs the formation of the XDH (alphabeta)(2) heterotetramer and, thus, insertion of Moco into the enzyme. The results reveal that the insertion of the different redox centers into R. capsulatus XDH takes place sequentially. Dimerization of two (alphabeta) dimers is necessary for insertion of sulfurated Moco into apo-XDH, the last step of XDH maturation.