Cooperativity in regulation of membrane protein function: phenomenological analysis of the effects of pH and phospholipids.

Interaction between membrane proteins and ligands plays a key role in governing a wide spectrum of cellular processes. These interactions can provide a cooperative-type regulation of protein function. A wide variety of proteins, including enzymes, channels, transporters, and receptors, displays cooperative behavior in their interactions with ligands. Moreover, the ligands involved encompass a vast diversity and include specific molecules or ions that bind to specific binding sites. In this review, our particular focus is on the interaction between integral membrane proteins and ligands that can present multiple "binding sites", such as protons or membrane phospholipids. The study of the interaction that protons or lipids have with membrane proteins often presents challenges for classical mechanistic modeling approaches. In this regard, we show that, like Hill's pioneering work on hemoglobin regulation, phenomenological modeling constitutes a powerful tool for capturing essential features of these systems.

Nonextensive realizations in interacting ion channels: Implications for mechano-electrical transducer mechanisms.

We propose a theoretical model to investigate the thermodynamics of single and coupled two-state ion channels, associated with mechanoelectrical transduction (MET) and hair cell biophysics. The modeling was based on the Tsallis nonextensive statistical mechanics. The choice for a nonextensive framework in modeling ion channels is encouraged on the fact that we take into account the presence of interactions or long-range correlations in the dynamics of single and coupled ion channels. However, the basic assumptions that support Boltzmann-Gibbs statistics, traditionally used to model ion channel dynamics, state that the system is formed by independent or weakly interacting elements. Despite being well studied in many biological systems, the literature has not yet addressed the study of both entropy and mutual information related to isolated or physically interacting pairs of MET channels. Inspired by hair cell biophysics, we show how the presence of nonextensivity, or subadditivity and superadditivity modulates the nonextensive entropy and mutual information as functions of stereocilia displacements. We also observe that the magnitude of the interaction between the two channels, given by a nonextensive parameter, influences the amplitude of the nonextensive joint entropy and mutual information as functions of the hair cell displacements. Finally, we show how nonextensivity regulates the current versus displacement curve for a single and a pair of interacting two-state channels. The present findings shed light on the thermodynamic process involved in the molecular mechanisms of the auditory system.

Thermodynamic and kinetic analysis of the LAO binding protein and its isolated domains reveal non-additivity in stability, folding and function.

Substrate-binding proteins (SBPs) are used by organisms from the three domains of life for transport and signalling. SBPs are composed of two domains that collectively trap ligands with high affinity and selectivity. To explore the role of the domains and the integrity of the hinge region between them in the function and conformation of SBPs, here, we describe the ligand binding, conformational stability and folding kinetics of the Lysine Arginine Ornithine (LAO) binding protein from Salmonella thiphimurium and constructs corresponding to its two independent domains. LAO is a class II SBP formed by a continuous and a discontinuous domain. Contrary to the expected behaviour based on their connectivity, the discontinuous domain shows a stable native-like structure that binds l-arginine with moderate affinity, whereas the continuous domain is barely stable and shows no detectable ligand binding. Regarding folding kinetics, studies of the entire protein revealed the presence of at least two intermediates. While the unfolding and refolding of the continuous domain exhibited only a single intermediate and simpler and faster kinetics than LAO, the folding mechanism of the discontinuous domain was complex and involved multiple intermediates. These findings suggest that in the complete protein the continuous domain nucleates folding and that its presence funnels the folding of the discontinuous domain avoiding nonproductive interactions. The strong dependence of the function, stability and folding pathway of the lobes on their covalent association is most likely the result of the coevolution of both domains as a single unit.

Substrate binding in the allosteric site mimics homotropic cooperativity in the SIS-fold glucosamine-6-phosphate deaminases.

Glucosamine-6-phosphate (GlcN6P) deaminases from Escherichia coli (EcNagBI) and Shewanella denitrificans (SdNagBII) are special examples of what constitute nonhomologous isofunctional enzymes due to their convergence, not only in catalysis, but also in cooperativity and allosteric properties. Additionally, we found that the sigmoidal kinetics of SdNagBII cannot be explained by the existing models of homotropic activation. This study describes the regulatory mechanism of SdNagBII using enzyme kinetics, isothermal titration calorimetry (ITC), and X-ray crystallography. ITC experiments revealed two different binding sites with distinctive thermodynamic signatures: a single binding site per monomer for the allosteric activator N-acetylglucosamine 6-phosphate (GlcNAc6P) and two binding sites per monomer for the transition-state analog 2-amino-2-deoxy-D-glucitol 6-phosphate (GlcNol6P). Crystallographic data demonstrated the existence of an unusual allosteric site that can bind both GlcNAc6P and GlcNol6P, implying that the homotropic activation of this enzyme arises from the occupation of the allosteric site by the substrate. In this work we describe the presence of this novel allosteric site in the SIS-fold deaminases, which is responsible for the homotropic and heterotropic activation of SdNagBII by GlcN6P and GlcNAc6P, respectively. This study unveils an original mechanism to generate a high degree of homotropic activation in SdNagBII, mimicking the allosteric and cooperative properties of hexameric EcNagBI but with a reduced number of subunits.

Spectroscopic analysis of coenzyme binding to betaine aldehyde dehydrogenase dependent on potassium.

Glycine betaine is the main osmolyte synthesized and accumulated in mammalian renal cells. Glycine betaine synthesis is catalyzed by the enzyme betaine aldehyde dehydrogenase (BADH) using NAD+ as the coenzyme. Previous studies have shown that porcine kidney betaine aldehyde dehydrogenase (pkBADH) binds NAD+ with different affinities at each active site and that the binding is K+ dependent. The objective of this work was to analyze the changes in the pkBADH secondary and tertiary structure resulting from variable concentrations of NAD+ and the role played by K+ . Intrinsic fluorescence studies were carried out at fixed-variable concentrations of K+ and titrating the enzyme with varying concentrations of NAD+ . Fluorescence analysis showed a shift of the maximum emission towards red as the concentration of K+ was increased. Changes in the exposure of tryptophan located near the NAD+ binding site were found when the enzyme was titrated with NAD+ in the presence of potassium. Fluorescence data analysis showed that the K+ presence promoted static quenching that facilitated the pkBADH-NAD+ complex formation. DC data analysis showed that binding of K+ to the enzyme caused changes in the α-helix content of 4% and 12% in the presence of 25 mM and 100 mM K+ , respectively. The presence of K+ during NAD+ binding to pkBADH increased the thermal stability of the complex. These results indicated that K+ facilitated the pkBADH-NAD+ complex formation and suggested that K+ caused small changes in secondary and tertiary structures that could influence the active site conformation.

Structure and activation mechanism of the human liver-type glutaminase GLS2.

Cancer cells exhibit an altered metabolic phenotype, consuming higher levels of the amino acid glutamine. This metabolic reprogramming depends on increased mitochondrial glutaminase activity to convert glutamine to glutamate, an essential precursor for bioenergetic and biosynthetic processes in cells. Mammals encode the kidney-type (GLS) and liver-type (GLS2) glutaminase isozymes. GLS is overexpressed in cancer and associated with enhanced malignancy. On the other hand, GLS2 is either a tumor suppressor or an oncogene, depending on the tumor type. The GLS structure and activation mechanism are well known, while the structural determinants for GLS2 activation remain elusive. Here, we describe the structure of the human glutaminase domain of GLS2, followed by the functional characterization of the residues critical for its activity. Increasing concentrations of GLS2 lead to tetramer stabilization, a process enhanced by phosphate. In GLS2, the so-called "lid loop" is in a rigid open conformation, which may be related to its higher affinity for phosphate and lower affinity for glutamine; hence, it has lower glutaminase activity than GLS. The lower affinity of GLS2 for glutamine is also related to its less electropositive catalytic site than GLS, as indicated by a Thr225Lys substitution within the catalytic site decreasing the GLS2 glutamine concentration corresponding to half-maximal velocity (K0.5). Finally, we show that the Lys253Ala substitution (corresponding to the Lys320Ala in the GLS "activation" loop, formerly known as the "gating" loop) renders a highly active protein in stable tetrameric form. We conclude that the "activation" loop, a known target for GLS inhibition, may also be a drug target for GLS2.

Interactions in Model Ionic Dyads and Triads Containing Tetrel Atoms.

The interactions in model ionic YTX3···Z (Y = NC, F, Cl, Br; X = F, Cl, Br, Z = F-, Cl-, Br-, Li+) dyads containing the tetrel atoms, T = C, Si, Ge, were studied using ab initio computational methods, including an energy decomposition analysis, which found that the YTX3 molecules were stabilized by both anions (via tetrel bonding) and cations (via polarization). For the tetrel-bonded dyads, both the electrostatic and polarization forces make comparable contributions to the binding in the C-containing dyads, whereas, electrostatic forces are by far the largest contributor to the binding in the Si- and Ge-containing analogues. Model metastable Li+···NCTCl3···F- (T = C, Si, Ge) triads were found to be lower in energy than the combined energy of the Li+ + NCTCl3 + F- fragments. The pair energies and cooperative energies for these highly polar triads were also computed and discussed.

Insights on the participation of Glu256 and Asp204 in the oligomeric structure and cooperative effects of human arginase type I.

Arginase (EC 3.5.3.1) catalyzes the hydrolysis of L-arginine to L-ornithine and urea, and requires a bivalent cation, especially Mn2+ for its catalytic activity. It is a component of the urea cycle and regulates the intracellular levels of l-arginine, which makes the arginase a target for treatment of vascular diseases and asthma. Mammalian arginases contain an unusual S-shaped motif located at the intermonomeric interface. Until now, the studies were limited to structural role of the motif. Then, our interest was focused on functional aspects and our hypothesis has been that the motif is essential for maintain the oligomeric state, having Arg308 as a central axis. Previously, we have shown that the R308A mutant is monomeric and re-associates to the trimeric-cooperative state in the presence of low concentrations of guanidine chloride. We have now mutated Asp204 that interacts with Arg308 in the neighbor subunit, and also we mutated Glu256, proposed as important for oligomerization. Concretely, the human arginase I mutants D204A, D204E, E256A, E256Q and E256D were generated and examined. No differences were observed in the kinetic parameters at pH 9.5 or in tryptophan fluorescence. However, the D204A and E256Q variants were monomeric. On the other hand, D204E and E256D proved to be trimeric and kinetically cooperative at pH 7.5, whereas hyperbolic kinetics was exhibited by E256A, also trimeric. The results obtained strongly support the importance of the interaction between Arg255 and Glu256 in the cooperative properties of arginase, and Asp204 would be relevant to maintain the oligomeric state through salt bridges with Arg255 and Arg308.

Physiological Role of Glutamate Dehydrogenase in Cancer Cells.

NH 4 + increased growth rates and final densities of several human metastatic cancer cells. To assess whether glutamate dehydrogenase (GDH) in cancer cells may catalyze the reverse reaction of NH 4 + fixation, its covalent regulation and kinetic parameters were determined under near-physiological conditions. Increased total protein and phosphorylation were attained in NH 4 + -supplemented metastatic cells, but total cell GDH activity was unchanged. Higher V max values for the GDH reverse reaction vs. forward reaction in both isolated hepatoma (HepM) and liver mitochondria [rat liver mitochondria (RLM)] favored an NH 4 + -fixing role. GDH sigmoidal kinetics with NH 4 + , ADP, and leucine fitted to Hill equation showed n H values of 2 to 3. However, the K 0.5 values for NH 4 + were over 20 mM, questioning the physiological relevance of the GDH reverse reaction, because intracellular NH 4 + in tumors is 1 to 5 mM. In contrast, data fitting to the Monod-Wyman-Changeux (MWC) model revealed lower K m values for NH 4 + , of 6 to 12 mM. In silico analysis made with MWC equation, and using physiological concentrations of substrates and modulators, predicted GDH N-fixing activity in cancer cells. Therefore, together with its thermodynamic feasibility, GDH may reach rates for its reverse, NH 4 + -fixing reaction that are compatible with an anabolic role for supporting growth of cancer cells.

4'-Hydroxy-6,7-methylenedioxy-3-methoxyflavone: A novel flavonoid from Dulacia egleri with potential inhibitory activity against cathepsins B and L.

A new flavone, 4'-hydroxy-6,7-methylenedioxy-3-methoxyflavone 1, and two other nucleosides, ribavirin 2 and adenosine 3, were isolated from the leaves of Dulacia egleri. The nucleosides were identified by spectroscopic techniques (1D, 2D-NMR) while the structure of the flavonoid was established by 1D, 2D-NMR analysis, including HRESIMS data. The results obtained in the biological assays showed that the compound 1 was able to inhibit cathepsins B and L with IC50 of 14.88 ±â€¯0.18 µM and 3.19 ±â€¯0.07 µM, respectively. The mechanism of inhibition for both enzymes were determined showing to be competitive at cathepsin B with Ki = 12.8 ±â€¯0.6 µM and non-linear non-competitive with positive cooperativity inhibition at cathepsin L with Ki = 322 ±â€¯33 µM, αKi = 133 ±â€¯15 µM, ßKi = 5.14 ±â€¯0.41 µM and γKi = 13.2 ±â€¯13 µM.

Cooperativity in proton sensing by PIP aquaporins.

One of the most intriguing properties of plasma membrane intrinsic protein (PIP) aquaporins (AQPs) is their ability to modulate water transport by sensing different levels of intracellular pH through the assembly of homo- and heterotetrameric molecular species in the plasma membrane. In this work, using a phenomenological modeling approach, we demonstrate that cooperativity in PIP biological response cannot be directly attributed to a cooperative proton binding, as it is usually considered, since it could also be the consequence of a cooperative conformation transition between open and closed states of the channel. Moreover, our results show that, when mixed populations of homo- and heterotetrameric PIP channels are coexpressed in the plasma membrane of the same cell, the observed decrease in the degree of positive cooperativity would result from the simultaneous presence of molecular species with different levels of proton sensing. Indeed, the random mixing between different PIP paralogues as subunits in a single tetramer, plus the possibility of mixed populations of homo- and heterotetrameric PIP channels widen the spectrum of cooperative responses of a cell membrane. Our approach offers a deep understanding of cooperative transport of AQP channels, as members of a multiprotein family where the relevant proton binding sites of each member have not been clearly elucidated yet.

Structural studies of glyceraldehyde-3-phosphate dehydrogenase from Naegleria gruberi, the first one from phylum Percolozoa.

Naegleria gruberi is a free life amoeba believed to have more than one billion years of existence; it is not pathogenic and had its genome sequenced, which revealed a high complexity in the metabolic pathways. This paper presents the experimental structure of GAPDH from N. gruberi, the first one belonging to the phylum Percolozoa, comparisons to structures from various species and molecular dynamics studies of some particular features. The final refined structure presents Rcryst = 15.54% and Rfree = 19.84%. The catalytic domain formed by residues 134 to 313 is highly conserved, as expected, with the exception of Asn145, present only in NgGAPDH, while the other GAPDHs present either Ser or Thr on the corresponding position. Molecular dynamics analysis revealed that Asn145 has correlated motions with residues Ala123, Thr125 and Pro126 that belong to what was called "bonded loop". NgGAPDH residue Met35 presents an extended side chain, closer to the cofactor adenine ring than corresponding (different) residues and conformations found in some parasitic protozoa and the human GAPDHs. The enzyme was previously reported to present positive cooperativity, which is hypothesized to be related to certain atom distances.

Cooperative RNA Recognition by a Viral Transcription Antiterminator.

RNA transcription of mononegavirales decreases gradually from the 3' leader promoter toward the 5' end of the genome, due to a decay in polymerase processivity. In the respiratory syncytial virus and metapneumovirus, the M2-1 protein ensures transcription anti-termination. Despite being a homotetramer, respiratory syncytial virus M2-1 binds two molecules of RNA of 13mer or longer per tetramer, and temperature-sensitive secondary structure in the RNA ligand is unfolded by stoichiometric interaction with M2-1. Fine quantitative analysis shows positive cooperativity, indicative of conformational asymmetry in the tetramer. RNA binds to M2-1 through a fast bimolecular association followed by slow rearrangements corresponding to an induced-fit mechanism, providing a sequential description of the time events of cooperativity. The first binding event of half of the RNA molecule to one of the sites increases the affinity of the second binding event on the adjacent contacting protomer by 15-fold, product of increased effective concentration caused by the entropic link. This mechanism allows for high-affinity binding with an otherwise relaxed sequence specificity, and instead suggests a yet undefined structural recognition signature in the RNA for modulating gene transcription. This work provides a basis for an essential event for understanding transcription antitermination in pneumoviruses and its counterpart Ebola virus VP30.

Specificity of peptidases secreted by filamentous fungi.

Peptidases are enzymes that cleave peptide bonds, yielding proteins and peptides. Enzymes in this class also perform several other functions, regulating the activation or inactivation of target substrates via proteolysis. Owing to these functions, peptidases have been extensively used in industrial and biotechnological applications. Given their potential functions, it is important to optimize the use of these enzymes, which requires determination of the specificity of each peptidase. The peptidase specificity must be taken into account in choosing a peptidase to catalyze the available protein source within the desired application. The specificity of a peptidase defines the profile of enzyme-substrate interactions, and for this the catalytic site and the arrangement of the amino acid residues involved in peptide bond cleavage need to be known. The catalytic sites of peptidases may be composed of several subsites that interact with amino acid residues for proteolysis. Filamentous fungi produce peptidases with varying specificity, and here we provide a review of those reported to date and their potential applications.

The oxygen-binding properties of hemocyanin from the mollusk Concholepas concholepas.

Hemocyanins have highly conserved copper-containing active sites that bind oxygen. However, structural differences among the hemocyanins of various mollusks may affect their physicochemical properties. Here, we studied the oxygen-binding cooperativity and affinity of Concholepas concholepas hemocyanin (CCH) and its two isolated subunits over a wide range of temperatures and pH values. Considering the differences in the quaternary structures of CCH and keyhole limpet hemocyanin (KLH), we hypothesized that the heterodidecameric CCH has different oxygen-binding parameters than the homodidecameric KLH. A novel modification of the polarographic method was applied in which rat liver submitochondrial particles containing cytochrome c oxidase were introduced to totally deplete oxygen of the test solution using ascorbate as the electron donor. This method was both sensitive and reproducible. The results showed that CCH, like other hemocyanins, exhibits cooperativity, showing an inverse relationship between the oxygen-binding parameters and temperature. According to their Hill coefficients, KLH has greater cooperativity than CCH at physiological pH; however, CCH is less sensitive to pH changes than KLH. Appreciable differences in binding behavior were found between the CCH subunits: the cooperativity of CCH-A was not only almost double that of CCH-B, but it was also slightly superior to that of CCH, thus suggesting that the oxygen-binding domains of the CCH subunits are different in their primary structure. Collectively, these data suggest that CCH-A is the main oxygen-binding domain in CCH; CCH-B may play a more structural role, perhaps utilizing its surprising predisposition to form tubular polymers, unlike CCH-A, as demonstrated here using electron microscopy.

Benchmark, DFT assessments, cooperativity, and energy decomposition analysis of the hydrogen bonds in HCN/HNC oligomeric complexes.

Hydrogen cyanide (HCN) and its tautomer hydrogen isocyanide (HNC) are relevant for extraterrestrial chemistry and possible relation to the origin of biomolecules. Several processes and reactions involving these molecules depend on their intermolecular interactions that can lead to aggregates and liquids especially due to the hydrogen bonds. It is thus important to comprehend, to describe, and to quantify their hydrogen bonds, mainly their nature and the cooperativity effects. A systematic study of all linear complexes up to pentamers of HCN and HNC is presented. CCSD(T)/CBS energy calculations, with and without basis set superposition error (BSSE) corrections for energies and geometries, provided a suitable set of benchmarks. Approximated methods based on the density functional theory (DFT) such as BP86, PBE, TPSS, B3LYP, CAM-B3LYP with and without dispersion corrections and long-range corrections, were assessed to describe the interaction energies and cooperativity effects. These assessments are relevant to select DFT functionals for liquid simulations. Energy decomposition analysis was performed at the PBE/STO-TZ2P level and provided insights into the nature of the hydrogen bonds, which are dominated by the electrostatic component. In addition, several linear relationships between the various energy components and the interaction energy were obtained. The cooperativity energy was also found to be practically linear with respect to the interaction energy, which may be relevant for designing and/or correcting empirical force fields. Graphical Abstract Hydrogen bonds in HCN/HNC oligomeric complexesᅟ.

A novel mechanism of functional cooperativity regulation by thiol redox status in a dimeric inorganic pyrophosphatase.

BACKGROUND: Inorganic PPases are essential metal-dependent enzymes that convert pyrophosphate into orthophosphate. This reaction is quite exergonic and provides a thermodynamic advantage for many ATP-driven biosynthetic reactions. We have previously demonstrated that cytosolic PPase from R. microplus embryos is an atypical Family I PPase. Here, we explored the functional role of the cysteine residues located at the homodimer interface, its redox sensitivity, as well as structural and kinetic parameters related to thiol redox status. METHODS: In this work, we used prokaryotic expression system for recombinant protein overexpression, biochemical approaches to assess kinetic parameters, ticks embryos and computational approaches to analyze and predict critical amino acids as well as physicochemical properties at the homodimer interface. RESULTS: Cysteine 339, located at the homodimer interface, was found to play an important role in stabilizing a functional cooperativity between the two catalytic sites, as indicated by kinetics and Hill coefficient analyses of the WT-rBmPPase. WT-rBmPPase activity was up-regulated by physiological antioxidant molecules such as reduced glutathione and ascorbic acid. On the other hand, hydrogen peroxide at physiological concentrations decreased the affinity of WT-rBmPPase for its substrate (PPi), probably by inducing disulfide bridge formation. CONCLUSIONS: Our results provide a new angle in understanding redox control by disulfide bonds formation in enzymes from hematophagous arthropods. The reversibility of the down-regulation is dependent on hydrophobic interactions at the dimer interface. GENERAL SIGNIFICANCE: This study is the first report on a soluble PPase where dimeric cooperativity is regulated by a redox mechanism, according to cysteine redox status.

Influence of Metal, Ligand and Solvent on Supramolecular Polymerizations with Transition-Metal Compounds: A Theoretical Study.

The nature of intermolecular interactions governing supramolecular polymerizations is very important for controlling their cooperativity. In order to address this problem, supramolecular columns made of PtII and PdII complexes of oligo(phenylene ethynylene)-based pyridine (OPE) and tetrazolylpyridine ligands (TEP) were investigated through the dispersion-corrected PM6 method. Aromatic, CH-π, M-Cl and metallophilic interactions helped stabilize the supramolecules studied, and their geometries and associated cooperativities were in excellent agreement with experimental data. The OPE ligand and/or the presence of PtII led to stronger metallophilic interactions and also to cooperative supramolecular polymerizations, which clearly suggests that metallophilic interactions are a key factor for controlling cooperativity. The results indicate that sequential monomer addition is in general less spontaneous than the combination of two larger preformed stacks. The present theoretical investigations contribute to the further understanding of the relation between the thermodynamics of supramolecular polymerizations and the nature of different synthons.

Evolution of the hydrogen-bonding motif in the melamine-cyanuric acid co-crystal: a topological study.

The melamine (M)/cyanuric acid (CA) supramolecular system is perhaps one of the most exploited in the field of self-assembly because of the high complementarity of the components. However, it is necessary to investigate further the factors involved in the assembly process. In this study, we analyzed a set of 13 M n /CA m clusters (with n , m = 1, 2, 3), taken from crystallographic data, to characterize the nature of the hydrogen bonds involved in the self-assembly of these components as well as to provide greater understanding of the phenomenon. The calculations were performed at the B3LYP/6-311++G(d,p) and ω-B97XD (single point) levels of theory, and the interactions were analyzed within the framework of the quantum theory of atoms in molecules and by means of molecular electrostatic potential maps. Our results show that the stablest structure is the rosette-type motif and the aggregation mechanism is governed by a combination of cooperative and anticooperative effects. Our topological results explain the polymorphism in the self-assembly of coadsorbed monolayers of M and CA. Graphical abstract The aggregation steps of the melamine-cyanuric co-crystal is driven by a hydrogen-bonded network which is governed by a complex combination of cooperative and anticooperative effects.

The mechanistic study of Leishmania major dihydro-orotate dehydrogenase based on steady- and pre-steady-state kinetic analysis.

Leishmania major dihydro-orotate dehydrogenase (DHODHLm) has been considered as a potential therapeutic target against leishmaniasis. DHODHLm, a member of class 1A DHODH, oxidizes dihydro-orotate (DHO) to orotate (ORO) during pyrimidine biosynthesis using fumarate (FUM) as the oxidizing substrate. In the present study, the chemistry of reduction and reoxidation of the flavin mononucleotide (FMN) cofactor in DHODHLm was examined by steady- and pre-steady state kinetics under both aerobic and anaerobic environments. Our results provide for the first time the experimental evidence of co-operative behaviour in class 1A DHODH regulated by DHO binding and reveal that the initial reductive flavin half-reaction follows a mechanism with two steps. The first step is consistent with FMN reduction and shows a hyperbolic dependence on the DHO concentration with a limiting rate (kred) of 110±6 s(-1) and a K(DHO) d of 180±27 µM. Dissociation of the reduced flavin-ORO complex corresponds to the second step, with a limiting rate of 6 s(-1). In the oxidative half-reaction, the oxygen-sensitive reoxidation of the reduced FMN cofactor of DHODHLm by FUM exhibited a hyperbolic saturation profile dependent on FUM concentration allowing estimation of K(FUM) d and the limiting rate (kreox) of 258±53 µM and 35±2 s(-1), respectively. Comparison between steady- and pre-steady-state parameters together with studies of interaction for DHODHLm with both ORO and succinate (SUC), suggests that ORO release is the rate-limiting step in overall catalysis. Our results provide evidence of mechanistic differences between class 1A and class 2 individual half-reactions to be exploited for the development of selective inhibitors.