Novel EPR signals associated with FeMoco centres of MoFe protein in MgADP-inhibited turnover of nitrogenase.

Two novel electron paramagnetic resonance (EPR) signals arising from the [1Mo-7Fe-9S-homocitrate] (FeMoco) centres of MoFe protein of Klebsiella pneumoniae nitrogenase (Kp1) were observed following turnover under MgATP-limited conditions. The combination of the nitrogenase Fe protein of Clostridium pasteurianum showed similar signals. The accumulation of MgADP under these conditions causes the normal EPR signal of dithionite-reduced Kp1 (with g=4.3, 3.6, 2.01) to be slowly converted to novel signals with g=4.74, 3.32, 2.00 and g=4.58, 3.50, 1.99. These signals do not form in incubation of protein mixtures containing only MgADP, thus they may be associated with trapped intermediates of the catalytic cycle.

Oxalate decarboxylase requires manganese and dioxygen for activity. Overexpression and characterization of Bacillus subtilis YvrK and YoaN.

The Bacillus subtilis oxalate decarboxylase (EC ), YvrK, converts oxalate to formate and CO(2). YvrK and the related hypothetical proteins YoaN and YxaG from B. subtilis have been successfully overexpressed in Escherichia coli. Recombinant YvrK and YoaN were found to be soluble enzymes with oxalate decarboxylase activity only when expressed in the presence of manganese salts. No enzyme activity has yet been detected for YxaG, which was expressed as a soluble protein without the requirement for manganese salts. YvrK and YoaN were found to catalyze minor side reactions: oxalate oxidation to produce H(2)O(2); and oxalate-dependent, H(2)O(2)-independent dye oxidations. The oxalate decarboxylase activity of purified YvrK was O(2)-dependent. YvrK was found to contain between 0.86 and 1.14 atoms of manganese/subunit. EPR spectroscopy showed that the metal ion was predominantly but not exclusively in the Mn(II) oxidation state. The hyperfine coupling constant (A = 9.5 millitesla) of the main g = 2 signal was consistent with oxygen and nitrogen ligands with hexacoordinate geometry. The structure of YvrK was modeled on the basis of homology with oxalate oxidase, canavalin, and phaseolin, and its hexameric oligomerization was predicted by analogy with proglycinin and homogentisate 1,2-dioxygenase. Although YvrK possesses two potential active sites, only one could be fully occupied by manganese. The possibility that the C-terminal domain active site has no manganese bound and is buried in an intersubunit interface within the hexameric enzyme is discussed. A mechanism for oxalate decarboxylation is proposed, in which both Mn(II) and O(2) are cofactors that act together as a two-electron sink during catalysis.

Long-range interactions between the Fe protein binding sites of the MoFe protein of nitrogenase.

We report the properties and reactivity of the catalytically active heterologous nitrogenase formed between the Fe protein from Clostridium pasteurianum (Cp2) and the MoFe protein from Klebsiella pneumoniae (Kp1). Under turnover conditions, in the presence of MgATP, a stable 2:1 (Cp2)2Kp1 electron transfer complex is formed, in which the [4Fe-4S]+ centre of Cp2 is protected from chelation by alpha,alpha'-bipyridyl. However, the two Fe protein-binding sites on Kp1 are not equivalent, since a 1:1 Cp2.Kp1 complex was isolated by gel filtration. The non-equivalence of the Fe protein binding sites was also indicated by the inhibition pattern of Klebsiella nitrogenase by Cp2. The EPR spectrum of the isolated 1:1 Cp2.Kp1 complex showed an S=1/2 signal characteristic of dithionite-reduced Cp2 and signals with g values of 4.27, 3.73, 2.01 and 4.32, 3.63, 2.00 characteristic of the high- and low-pH forms of the FeMoco centre of Kp1, respectively. The unoccupied binding site of Kp1 of the isolated 1:1 Cp2Kp1 complex was shown to be catalytically fully functional in combination with Kp2. In contrast to homologous nitrogenases, which require MgATP for detectable rates of electron transfer from the Fe protein, stopped-flow kinetic studies revealed that electron transfer from Cp2 to Kp1 occurred in the absence of MgATP with a rate constant of 0.065 s(-1). Subsequently, a slower transient decrease and restoration of absorption in the electronic spectrum in the 500-700 nm region was observed. These changes corresponded with those in the intensity of the S=3/2 EPR signal of the FeMoco centres of Kp1 and were consistent with the transient reduction of the FeMoco centre of Kp1 to an EPR-silent form, followed by restoration of the signal at longer reaction times. These changes were not associated with catalysis since no evolution of H2 was detectable.

Transition state complexes of the Klebsiella pneumoniae nitrogenase proteins. Spectroscopic properties of aluminium fluoride-stabilized and beryllium fluoride-stabilized MgADP complexes reveal conformational differences of the Fe protein.

Stable inactive 2 : 1 complexes of the Klebsiella pneumoniae nitrogenase components (Kp2/Kp1) were prepared with ADP or the fluorescent ADP analogue, 2'(3')-O-[N-methylanthraniloyl] ADP and AlF(4)(-) or BeF(3)(-) ions. By analogy with published crystallographic data [Schindelin et al. (1997) Nature 387, 370-376)], we suggest that the metal fluoride ions replaced phosphate at the two ATP-binding sites of the iron protein, Kp2. The beryllium (BeF(x)) and aluminium (AlF(4)(-)) containing complexes are proposed to correspond to the ATP-bound state and the hydrolytic transition states, respectively, by analogy with the equivalent complexes of myosin [Fisher et al. (1995) Biochemistry 34, 8960-8972]. (31)P NMR spectroscopy showed that during the initial stages of complex formation, MgADP bound to the complexed Kp2 in a manner similar to that reported for isolated Kp2. This process was followed by a second step that caused broadening of the (31)P NMR signals and, in the case of the AlF4- complex, slow hydrolysis of some of the excess ADP to AMP and inorganic phosphate. The purified BeFx complex contained 3.8 +/- 0.1 MgADP per mol Kp1. With the AlF(4)(-) complex, MgAMP and adenosine (from MgAMP hydrolysis) replaced part of the bound MgADP although four AlF(4)(-) ions were retained, demonstrating that full occupancy by MgADP is not required for the stability of the complex. The fluorescence emission maximum of 2'(3')-O-[N-methylanthraniloyl] ADP was blue-shifted by 6-8 nm in both metal fluoride complexes and polarization was 6-9 times that of the free analogue. The fluorescence yield of bound 2'(3')-O-[N-methylanthraniloyl] ADP was enhanced by 40% in the AlF(4)(-) complex relative to the solvent but no increase in fluorescence was observed in the BeFx complex. Resonance energy transfer from conserved tyrosine residues located in proximity to the Kp2 nucleotide-binding pocket was marked in the AlF(4)(-) complex but minimal in the BeFx fluoride complex, illustrating a clear conformational difference in the Fe protein of the two complexes. Our data indicate that complex formation during the nitrogenase catalytic cycle is a multistep process involving at least four conformational states of Kp2: similar to the free Fe protein; as initially complexed with detectable (31)P NMR; as detected in mature complexes with no detectable (31)P NMR; in the AlF(4)(-) complex in which an altered tyrosine interaction permits resonance energy transfer with 2'(3')-O-[N-methylanthraniloyl] ADP.

Mutagenesis and modelling of linoleate-binding to pea seed lipoxygenase.

We have produced a model to define the linoleate-binding pocket of pea 9/13-lipoxygenase and have validated it by the construction and characterization of eight point mutants. Three of the mutations reduced, to varying degrees, the catalytic centre activity (kcat) of the enzyme with linoleate. In two of the mutants, reductions in turnover were associated with changes in iron-coordination. Multiple sequence alignments of recombinant plant and mammalian lipoxygenases of known positional specificity, and the results from numerous other mutagenesis and modelling studies, have been combined to discuss the possible role of the mutated residues in pea 9/13-lipoxygenase catalysis. A new nomenclature for recombinant plant lipoxygenases based on positional specificity has subsequently been proposed. The null-effect of mutating pea 9/13-lipoxygenase at the equivalent residue to that which controlled dual positional specificity in cucumber 13/9-lipoxygenase, strongly suggests that the mechanisms controlling dual positional specificity in pea 9/13-lipoxygenase and cucumber 13/9-lipoxygenase are different. This was supported from modelling of another isoform of pea lipoxygenase, pea 13/9-lipoxygenase. Dual positional specificity in pea lipoxygenases is more likely to be determined by the degree of penetration of the methyl terminus of linoleate and the volume of the linoleate-binding pocket rather than substrate orientation. A single model for positional specificity, that has proved to be inappropriate for arachidonate-binding to mammalian 5-, 12- and 15-lipoxygenases, would appear to be true also for linoleate-binding to plant 9- and 13-lipoxygenases.

Probing a novel potato lipoxygenase with dual positional specificity reveals primary determinants of substrate binding and requirements for a surface hydrophobic loop and has implications for the role of lipoxygenases in tubers.

A new potato tuber lipoxygenase full-length cDNA sequence (lox1:St:2) has been isolated from potato tubers and used to express in Escherichia coli and characterize a novel recombinant lipoxygenase (potato 13/9-lipoxygenase). Like most plant lipoxygenases it produced carbonyl compounds from linoleate (the preferred substrate) and was purified in the Fe(II) (ferrous) state. Typical of other potato tuber lipoxygenases, it produced 5-HPETE [5(S)-hydroperoxy-(6E, 8Z, 11Z, 14Z)-eicosatetraenoic acid] from arachidonate. In contrast to any other potato tuber lipoxygenase, it exhibited dual positional specificity and produced roughly equimolar amounts of 13- and 9-hydroperoxides (or only a slight molar excess of 9-hydroperoxides) from linoleate. We have used a homology model of pea 9/13-lipoxygenase to superimpose and compare the linoleate-binding pockets of different potato lipoxygenases of known positional specificity. We then tested this model by using site-directed mutagenesis to identify some primary determinants of linoleate binding to potato 13/9-lipoxygenase and concluded that the mechanism determining positional specificity described for a cucumber lipoxygenase does not apply to potato 13/9-lipoxygenase. This supports our previous studies on pea seed lipoxygenases for the role of pocket volume rather than inverse orientation as a determinant of dual positional specificity in plant lipoxygenases. We have also used deletion mutagenesis to identify a critical role in catalysis for a surface hydrophobic loop in potato 13/9-lipoxygenase and speculate that this may control substrate access. Although potato 13/9-lipoxygenase represents only a minor isoform in tubers, such evidence for a single lipoxygenase species with dual positional specificity in tubers has implications for the proposed role of potato lipoxygenases in the plant.

Nucleotide binding by the nitrogenase Fe protein: a 31P NMR study of ADP and ATP interactions with the Fe protein of Klebsiella pneumoniae.

Investigation of the interaction of MgADP- and MgATP2- with the Fe protein of Klebsiella pneumoniae nitrogenase by 31P NMR showed that the adenine nucleotides are reversibly bound in slow exchange with free nucleotides. Dissociation of the MgADP--Fe protein complex was slow enough to enable its isolation by gel filtration, thus permitting the assignment of resonances to bound nucleotides. Spectra of ADP bound to Kp2 were similar to spectra of ADP bound to the myosin motor domain. Oxidative inactivation of a Kp2-MgADP- complex with excess ferricyanide ion eliminated exchange between bound and free ADP, indicating that the intact iron sulphur cluster, located 20 A from the binding sites, is required for the reversible binding of MgADP-. A change in conformation on controlled oxidation of Kp2 with indigocarmine increased the chemical shift of the beta phosphate resonance of bound MgADP-. Both oxidized and reduced conformers were observed transiently in the absence of dithionite. The 31P resonances of both the beta and gamma phosphates of bound MgATP2- indicated major changes in environment and labilization of both groups on binding to the Fe protein. Highly purified Kp2 slowly hydrolysed ATP, resulting in mixtures of bound nucleotides. Partial occupation of Kp2 MgATP2--binding sites (N=1.9+/-0.2, Kd=145 microM) in concentrated protein solutions was demonstrated by flow dialysis. Scatchard plots of data for bound and free ligand obtained after equilibration with Kp2 were linear and no co-operative interactions were detected. We conclude that MgADP- stabilizes the oxidized Fe protein conformer and this conformation in turn triggers the dissociation of the Fe protein from the MoFe protein in the rate-limiting step of the overall process of dinitrogen reduction.

Characterization of authentic recombinant pea-seed lipoxygenases with distinct properties and reaction mechanisms.

The two major isoforms of lipoxygenase (LOX-2 and LOX-3) from pea (Pisum sativum L. cv. Birte) seeds have been cloned and expressed from full-length cDNAs as soluble, active, non-fusion proteins in Escherichia coli. A comparison of both isoforms purified to apparent homogeneity from E. coli and pea seeds has confirmed the authenticity of the recombinant products and established the properties of the native enzymes. Despite 86% similarity at the amino acid sequence level, the enzymes have distinct properties. They have been characterized in terms of specific activity, Fe content, optimum pH, substrate and product specificity, apparent Km and Vmax for the preferred substrate, linoleic acid, and interfacial behaviour with linoleic acid. We have used this evidence, in addition to EPR spectroscopy of the hydroperoxide-activated enzymes and estimates of kcat/Km, to propose different reaction mechanisms for linoleic acid oxidation for the two isoforms. The differences relate primarily to carbonyl production from linoleic acid for which we propose a mechanism. This implicates the release of a peroxyl radical in an aerobic hydroperoxidase reaction, as the source of the carbonyl compounds formed by dismutation of the liberated peroxyl radical.

Novel keto acid formate-lyase and propionate kinase enzymes are components of an anaerobic pathway in Escherichia coli that degrades L-threonine to propionate.

An immunological analysis of an Escherichia coli strain unable to synthesize the main pyruvate formate-lyase enzyme Pfl revealed the existence of a weak, cross-reacting 85 kDa polypeptide that exhibited the characteristic oxygen-dependent fragmentation typical of a glycyl radical enzyme. Polypeptide fragmentation of this cross-reacting species was shown to be dependent on Pfl activase. Cloning and sequence analysis of the gene encoding this protein revealed that it coded for a new enzyme, termed TdcE, which has 82% identity with Pfl. On the basis of RNA analyses, the tdcE gene was shown to be part of a large operon that included the tdcABC genes, encoding an anaerobic threonine dehydratase, tdcD, coding for a propionate kinase, tdcF, the function of which is unknown, and the tdcG gene, which encodes a L-serine dehydratase. Expression of the tdcABCDEFG operon was strongly catabolite repressed. Enzyme studies showed that TdcE has both pyruvate formate-lyase and 2-ketobutyrate formate-lyase activity, whereas the TdcD protein is a new propionate/acetate kinase. By monitoring culture supernatants from various mutants using 1H nuclear magnetic resonance (NMR), we followed the anaerobic conversion of L-threonine to propionate. These studies confirmed that 2-ketobutyrate, the product of threonine deamination, is converted in vivo by TdcE to propionyl-CoA. These studies also revealed that Pfl and an as yet unidentified thiamine pyrophosphate-dependent enzyme(s) can perform this reaction. Double null mutants deficient in phosphotransacetylase (Pta) and acetate kinase (AckA) or AckA and TdcD were unable to metabolize threonine to propionate, indicating that propionyl-CoA and propionyl-phosphate are intermediates in the pathway and that ATP is generated during the conversion of propionyl-P to propionate by AckA or TdcD.

Covalent modification of nitrogenase MoFe protein by ADP.

MgADP- reacted with the nitrogenase molybdenum-iron (MoFe) protein of Klebsiella pneumoniae (Kp1) over a period of 2 h to yield a stable, catalytically active conjugate. The isolated protein exhibited a new, broad 31P NMR resonance at -1 p.p.m. lacking phosphorus J coupling. The adenine ring of [8-14C]ADP remained associated with the conjugate. A covalently bound nucleotide was identified as AMP by NMR and TLC. Extended dialysis of Kp1 against MgADP- resulted in further AMP binding at the protein surface. ADP was initially bound tightly to Kp1 at a site distinct from the AMP sites. ATP did not replace ADP. The time course of the formation of the Kp1-AMP was altered by the nitrogenase iron protein (Kp2) and was dependent on redox potential. Kp1-AMP was stable to concentration and oxidation with ferricyanide ion at -350 mV. Slow hydrolysis of Kp1-AMP over a period of 6 h yielded AMP and unaltered Kp1. The adenine ring of ADP exchanged with adenine of MgATP2- during reductant-limited turnover of nitrogenase under N2, indicating reversibility of ATP hydrolysis at 15 degrees C. [32P]Pi exchanged with the terminal phosphate group of both ADP and ATP on incubation with Kp1. 32P exchange and the catalytic activity of Kp1 were inhibited by a 20-fold molar excess of the lysine-modifying reagent, o-phthalaldehyde (OPT). Preincubation with MgADP- protected against OPT inactivation. Two potentially reactive lysine residues on the alpha chain of the MoFe protein near a putative hydrophobic docking site for the nitrogenase Fe protein are proposed as sites of OPT and nucleotide binding. Azotobacter vinelandii MoFe protein (Av1) also formed an AMP adduct but Kp2 did not. Catalase did not interact with ADP. The reactions of the nitrogenase MoFe protein with adenine nucleotides have no counterpart in known protein-nucleotide interactions.