A manganese(IV)/iron(IV) intermediate in assembly of the manganese(IV)/iron(III) cofactor of Chlamydia trachomatis ribonucleotide reductase

We recently showed that the class Ic ribonucleotide reductase from the human pathogen Chlamydia trachomatis uses a Mn(IV)/Fe(III) cofactor to generate protein and substrate radicals in its catalytic mechanism [Jiang, W., Yun, D., Saleh, L., Barr, E. W., Xing, G., Hoffart, L. M., Maslak, M.-A., Krebs, C., and Bollinger, J. M., Jr. (2007) Science 316, 1188-1191]. Here, we have dissected the mechanism of formation of this novel heterobinuclear redox cofactor from the Mn(II)/Fe(II) cluster and O2. An intermediate with a g = 2 EPR signal that shows hyperfine coupling to both 55Mn and 57Fe accumulates almost quantitatively in a second-order reaction between O2 and the reduced R2 complex. The otherwise slow decay of the intermediate to the active Mn(IV)/Fe(III)-R2 complex is accelerated by the presence of the one-electron reductant, ascorbate, implying that the intermediate is more oxidized than Mn(IV)/Fe(III). M?ssbauer spectra show that the intermediate contains a high-spin Fe(IV) center. Its chemical and spectroscopic properties establish that the intermediate is a Mn(IV)/Fe(IV)-R2 complex with an S = 1/2 electronic ground state arising from antiferromagnetic coupling between the Mn(IV) (S(Mn) = 3/2) and high-spin Fe(IV) (S(Fe) = 2) sites.     

Rapid and quantitative activation of Chlamydia trachomatis ribonucleotide reductase by hydrogen peroxide

We recently showed that the class Ic ribonucleotide reductase (RNR) from the human pathogen Chlamydia trachomatis ( Ct) uses a Mn (IV)/Fe (III) cofactor in its R2 subunit to initiate catalysis [Jiang, W., Yun, D., Saleh, L., Barr, E. W., Xing, G., Hoffart, L. M., Maslak, M.-A., Krebs, C., and Bollinger, J. M., Jr. (2007) Science 316, 1188-1191]. The Mn (IV) site of the novel cofactor functionally replaces the tyrosyl radical used by conventional class I RNRs to initiate substrate radical production. As a first step in evaluating the hypothesis that the use of the alternative cofactor could make the RNR more robust to reactive oxygen and nitrogen species [RO(N)S] produced by the host's immune system [H?gbom, M., Stenmark, P., Voevodskaya, N., McClarty, G., Gr?slund, A., and Nordlund, P. (2004) Science 305, 245-248], we have examined the reactivities of three stable redox states of the Mn/Fe cluster (Mn (II)/Fe (II), Mn (III)/Fe (III), and Mn (IV)/Fe (III)) toward hydrogen peroxide. Not only is the activity of the Mn (IV)/Fe (III)-R2 intermediate stable to prolonged (>1 h) incubations with as much as 5 mM H 2O 2, but both the fully reduced (Mn (II)/Fe (II)) and one-electron-reduced (Mn (III)/Fe (III)) forms of the protein are also efficiently activated by H 2O 2. The Mn (III)/Fe (III)-R2 species reacts with a second-order rate constant of 8 +/- 1 M (-1) s (-1) to yield the Mn (IV)/Fe (IV)-R2 intermediate previously observed in the reaction of Mn (II)/Fe (II)-R2 with O 2 [Jiang, W., Hoffart, L. M., Krebs, C., and Bollinger, J. M., Jr. (2007) Biochemistry 46, 8709-8716]. As previously observed, the intermediate decays by reduction of the Fe site to the active Mn (IV)/Fe (III)-R2 complex. The reaction of the Mn (II)/Fe (II)-R2 species with H 2O 2 proceeds in three resolved steps: sequential oxidation to Mn (III)/Fe (III)-R2 ( k = 1.7 +/- 0.3 mM (-1) s (-1)) and Mn (IV)/Fe (IV)-R2, followed by decay of the intermediate to the active Mn (IV)/Fe (III)-R2 product. The efficient reaction of both reduced forms with H 2O 2 contrasts with previous observations on the conventional class I RNR from Escherichia coli, which is efficiently converted from the fully reduced (Fe 2 (II/II)) to the "met" (Fe 2 (III/III)) form [Gerez, C., and Fontecave, M. (1992) Biochemistry 31, 780-786] but is then only very inefficiently converted from the met to the active (Fe 2 (III/III)-Y (*)) form [Sahlin, M., Sj?berg, B.-M., Backes, G., Loehr, T., and Sanders-Loehr, J. (1990) Biochem. Biophys. Res. Commun. 167, 813-818].     

The manganese/iron-carboxylate proteins: what is what, where are they, and what can the sequences tell us?

The manganese/iron-carboxylate proteins make up a recently discovered group of proteins that contain a heterodinuclear Mn/Fe redox cofactor. The chemical potential of the heterodinuclear metal site is just starting to be characterized, but available data suggest that it may have capabilities for similarly versatile chemistry as the extensively studied diiron-carboxylate cofactor. The presently identified members of the manganese/iron-carboxylate proteins are all sequence homologues of the radical-generating R2 subunit of class I ribonucleotide reductase, canonically a diiron protein. They are also commonly misannotated as such in databases. In spite of the sequence similarity, the manganese/iron-carboxylate proteins form at least two functionally distinct groups, radical-generating ribonucleotide reductase subunits and ligand-binding Mn/Fe proteins. Here, the presently available sequences for the manganese/iron-carboxylate proteins are gathered, grouped, and analyzed. The analysis provides sequence determinants that allow group identification of new sequences on the single-protein level. Key differences between the groups are mapped on the known representative structures, providing clues to the structural prerequisites for metal specificity, cofactor formation, and difference in function. The organisms that encode manganese/iron-carboxylate proteins are briefly discussed; their environmental preference suggests that the Mn/Fe heterodinuclear cofactor is preferred by extremophiles and pathogens with a particularly high relative presence in Archaea.     

Branched activation- and catalysis-specific pathways for electron relay to the manganese/iron cofactor in ribonucleotide reductase from Chlamydia trachomatis

A conventional class I (subclass a or b) ribonucleotide reductase (RNR) employs a tyrosyl radical (Y (*)) in its R2 subunit for reversible generation of a 3'-hydrogen-abstracting cysteine radical in its R1 subunit by proton-coupled electron transfer (PCET) through a network of aromatic amino acids spanning the two subunits. The class Ic RNR from the human pathogen Chlamydia trachomatis ( Ct) uses a Mn (IV)/Fe (III) cofactor (specifically, the Mn (IV) ion) in place of the Y (*) for radical initiation. Ct R2 is activated when its Mn (II)/Fe (II) form reacts with O 2 to generate a Mn (IV)/Fe (IV) intermediate, which decays by reduction of the Fe (IV) site to the active Mn (IV)/Fe (III) state. Here we show that the reduction step in this sequence is mediated by residue Y222. Substitution of Y222 with F retards the intrinsic decay of the Mn (IV)/Fe (IV) intermediate by approximately 10-fold and diminishes the ability of ascorbate to accelerate the decay by approximately 65-fold but has no detectable effect on the catalytic activity of the Mn (IV)/Fe (III)-R2 product. By contrast, substitution of Y338, the cognate of the subunit interfacial R2 residue in the R1 <--> R2 PCET pathway of the conventional class I RNRs [Y356 in Escherichia coli ( Ec) R2], has almost no effect on decay of the Mn (IV)/Fe (IV) intermediate but abolishes catalytic activity. Substitution of W51, the Ct R2 cognate of the cofactor-proximal R1 <--> R2 PCET pathway residue in the conventional class I RNRs (W48 in Ec R2), both retards reduction of the Mn (IV)/Fe (IV) intermediate and abolishes catalytic activity. These observations imply that Ct R2 has evolved branched pathways for electron relay to the cofactor during activation and catalysis. Other R2s predicted also to employ the Mn/Fe cofactor have Y or W (also competent for electron relay) aligning with Y222 of Ct R2. By contrast, many R2s known or expected to use the conventional Y (*)-based system have redox-inactive L or F residues at this position. Thus, the presence of branched activation- and catalysis-specific electron relay pathways may be functionally important uniquely in the Mn/Fe-dependent class Ic R2s.     

High-valent [MnFe] and [FeFe] cofactors in ribonucleotide reductases

Ribonucleotide reductases (RNRs) are essential for DNA synthesis in most organisms. In class-Ic RNR from Chlamydia trachomatis (Ct), a MnFe cofactor in subunit R2 forms the site required for enzyme activity, instead of an FeFe cofactor plus a redox-active tyrosine in class-Ia RNRs, for example in mouse (Mus musculus, Mm). For R2 proteins from Ct and Mm, either grown in the presence of, or reconstituted with Mn and Fe ions, structural and electronic properties of higher valence MnFe and FeFe sites were determined by X-ray absorption spectroscopy and complementary techniques, in combination with bond-valence-sum and density functional theory calculations. At least ten different cofactor species could be tentatively distinguished. In Ct R2, two different Mn(IV)Fe(III) site configurations were assigned either L(4)Mn(IV)(μO)(2)Fe(III)L(4) (metal-metal distance of ~2.75?, L = ligand) prevailing in metal-grown R2, or L(4)Mn(IV)(μO)(μOH)Fe(III)L(4) (~2.90?) dominating in metal-reconstituted R2. Specific spectroscopic features were attributed to an Fe(IV)Fe(III) site (~2.55?) with a L(4)Fe(IV)(μO)(2)Fe(III)L(3) core structure. Several Mn,Fe(III)Fe(III) (~2.9-3.1?) and Mn,Fe(III)Fe(II) species (~3.3-3.4?) likely showed 5-coordinated Mn(III) or Fe(III). Rapid X-ray photoreduction of iron and shorter metal-metal distances in the high-valent states suggested radiation-induced modifications in most crystal structures of R2. The actual configuration of the MnFe and FeFe cofactors seems to depend on assembly sequences, bound metal type, valence state, and previous catalytic activity involving subunit R1. In Ct R2, the protonation of a bridging oxide in the Mn(IV)(μO)(μOH)Fe(III) core may be important for preventing premature site reduction and initiation of the radical chemistry in R1.     

A Dynamic C-Terminal Segment in the Mycobacterium tuberculosis Mn/Fe R2lox Protein Can Adopt a Helical Structure with Possible Functional Consequences

Mycobacterium tuberculosis R2-like ligand-binding oxidase (MtR2lox) belongs to a recently discovered group of proteins that are homologous to the ribonucleotide reductase R2 proteins. MtR2lox carries a heterodinuclear Mn/Fe cofactor and, unlike R2 proteins, a large ligand-binding cavity. A unique tyrosine-valine cross link is also found in the vicinity of the active site. To date, all known structures of R2 and R2lox proteins show a disordered C-terminal segment. Here, we present two new crystal forms of MtR2lox, revealing an ordered helical C-terminal. The ability of alternating between an ordered and disordered state agrees well with bioinformatic analysis of the protein sequence. Interestingly, ordering of the C-terminal helix shields a large positively charged patch on the protein surface, potentially used for interaction with other cellular components. We hypothesize that the dynamic C-terminal segment may be involved in control of protein function in vivo.     

Structure of the manganese complex of photosystem II upon removal of the 33-kilodalton extrinsic protein: an X-ray absorption spectroscopy study

The structure of the Mn complex of photosystem II (PSII) was studied by X-ray absorption spectroscopy. Oxygen-evolving spinach PSII membranes containing 4-5 Mn/PSII were treated with 0.8 M CaCl2 to extract the 33-, 24-, and 16-kilodalton (kDa) extrinsic membrane proteins. Mn was not released by this treatment, but subsequent incubation at low Cl- concentration generated preparations containing 2 Mn/PSII. The Mn X-ray absorption K-edge spectrum of the CaCl2-washed preparation containing 4 Mn/PSII is very similar to spectrum of native PSII, indicating that the oxidation states and ligand symmetry of the Mn complex in these preparations are not significantly different. The Mn extended X-ray absorption fine structure (EXAFS) of CaCl2-washed PSII fits to a Mn neighbor at approximately 2.75 A and two shells of N or O at approximately 1.78 and approximately 1.92 A. These distances are similar to those we have previously reported for native PSII preparations [Yachandra, V. K., Guiles, R. D., McDermott, A. E., Cole, J. L., Britt, R. D., Dexheimer, S. L., Sauer, K., & Klein, M. P. (1987) Biochemistry (following paper in this issue)] and are indicative of an oxo-bridged Mn complex. Our results demonstrate that the structure of the Mn complex is largely unaffected by removal of 33-, 24-, and 16-kDa extrinsic proteins, do not provide ligands to Mn. The Mn K-edge spectrum of the CaCl2-washed sample containing 2 Mn/PSII has a dramatically altered shape, and the edge inflection point is shifted to lower energy. The position of the edge is consistent with a Mn oxidation state of +3.(ABSTRACT TRUNCATED AT 250 WORDS)     

A conserved tryptophan 457 modulates the kinetics and extent of N-hydroxy-L-arginine oxidation by inducible nitric-oxide synthase

In the oxygenase domain of mouse inducible nitric-oxide synthase (iNOSoxy), a conserved tryptophan residue, Trp-457, regulates the kinetics and extent of l-Arg oxidation to N(omega)-hydroxy-l-arginine (NOHA) by controlling electron transfer between bound (6R)-tetrahydrobiopterin (H(4)B) cofactor and the enzyme heme Fe(II)O(2) intermediate (Wang, Z. Q., Wei, C. C., Ghosh, S., Meade, A. L., Hemann, C., Hille, R., and Stuehr, D. J. (2001) Biochemistry 40, 12819-12825). To investigate whether NOHA oxidation to citrulline and nitric oxide (NO) is regulated by a similar mechanism, we performed single turnover reactions with wild type iNOSoxy and mutants W457F and W457A. Ferrous proteins containing NOHA plus H(4)B or NOHA plus 7,8-dihydrobiopterin (H(2)B), were mixed with O(2)-containing buffer, and then heme spectral transitions and product formation were followed versus time. All three proteins formed a Fe(II)O(2) intermediate with identical spectral characteristics. In wild type, H(4)B increased the disappearance rate of the Fe(II)O(2) intermediate relative to H(2)B, and its disappearance was coupled to the formation of a Fe(III)NO immediate product prior to formation of ferric enzyme. In W457F and W457A, the disappearance rate of the Fe(II)O(2) intermediate was slower than in wild type and took place without detectable build-up of the heme Fe(III)NO immediate product. Rates of Fe(II)O(2) disappearance correlated with rates of citrulline formation in all three proteins, and reactions containing H(4)B formed 1.0, 0.54, and 0.38 citrulline/heme in wild type, W457F, and W457A iNOSoxy, respectively. Thus, Trp-457 modulates the kinetics of NOHA oxidation by iNOSoxy, and this is important for determining the extent of citrulline and NO formation. Our findings support a redox role for H(4)B during NOHA oxidation to NO by iNOSoxy.     

Evidence for N coordination to Fe in the [2Fe-2S] clusters of Thermus Rieske protein and phthalate dioxygenase from Pseudomonas

Rieske-type iron/sulfur proteins and several NADH-dependent oxygenases contain Fe/S clusters with similar spectral and magnetic properties. Purified Rieske iron/sulfur protein from Thermus thermophilus contains two apparently identical [2Fe-2S] clusters in a polypeptide having only four cysteine residues, and it has been proposed that each Fe/S cluster is coordinated to two cysteine S-atoms and to an unknown number of other non-sulfur atoms (Fee, J. A., Findling, K. L., Yoshida, T., Hille, R., Tarr, G. E., Hearshen, D. O., Dunham, W. R., Day, E. P., Kent, T. A., and Munck, E. (1984) J. Biol. Chem. 259, 124-133). We have examined the Rieske protein from Thermus and the phthalate dioxygenase from Pseudomonas cepacia with electron nuclear double resonance (ENDOR) and pulsed EPR methods and report here evidence for the direct coordination of nitrogenous ligands to the Fe/S clusters in these proteins. The electron nuclear double resonance signals arising from 14N have been interpreted in terms of a strongly coupled ligand with AN = approximately 26-28 MHz and a weakly coupled ligand with AN = approximately 9 MHz. The pulsed EPR spectrum shows a rich pattern of lines in the Fourier transformed data having peaks in the range of 0.8 to 6.7 MHz. The lower frequency resonances are tentatively associated with coupling of the unpaired spin to the remote N-atoms of coordinated imidazole rings.     

A stable FeIII-FeIV replacement of tyrosyl radical in a class I ribonucleotide reductase

Ribonucleotide reductase (RNR) of Chlamydia trachomatis is a class I RNR enzyme composed of two homodimeric components, proteins R1 and R2. In class I RNR, R1 has the substrate binding site, whereas R2 has a diferric site and normally in its active form a stable tyrosyl free radical. C. trachomatis RNR is unusual, because its R2 component has a phenylalanine in the place of the radical carrier tyrosine. Replacing the tyrosyl radical, a paramagnetic Fe(III)-Fe(IV) species (species X, normally a transient intermediate in the process leading to radical formation) may provide the oxidation equivalent needed to start the catalytic process via long range electron transfer from the active site in R1. Here EPR spectroscopy shows that in C. trachomatis RNR, species X can become essentially stable when formed in a complete RNR (R1/R2/substrate) complex, adding further weight to the possible role of this species X in the catalytic reaction.     

M?ssbauer studies of solid thionin-oxidized MoFe protein of nitrogenase

Recently Hagen et al. (Hagen, W. R., Wassink, H., Eady, R. R., Smith, B. E., and Haaker, H. (1987) Eur. J. Biochem. 169, 457-465) reported the observation of S = 7/2 EPR signals for thionin-oxidized nitrogenase MoFe protein. Here we have studied the protein from Azotobacter vinelandii and Klebsiella pneumoniae with M?ssbauer and EPR spectroscopies, with the following results: when the MoFe protein is oxidized by addition of stoichiometric amounts (6-8 equivalents) of dissolved thionin, the well characterized P-cluster state Pox results. Pox has an as yet undetermined, but half-integer electronic spin; however, the state is EPR-silent. In contrast, oxidation by addition of a large excess of solid thionin powder, the method used by Hagen et al., yields mixtures with variable proportions of two oxidized P-cluster forms, namely the familiar Pox and the new state Pox(S = 7/2) observed by Hagen et al. The M?ssbauer data suggest that Pox and Pox(S = 7/2) are isoelectronic. The two states, however, have distinct electronic structures; the M?ssbauer spectra of Pox exhibit the characteristic trapped-valence Fe2+ site, whereas the spectra of Pox(S = 7/2) lack this feature. Hagen et al. have proposed two new P-cluster models. We conclude that one of the models is incompatible with the M?ssbauer data and that the basic assumptions of the other model are not supported by the available data. Finally, the M?ssbauer data show that either oxidation method puts the cofactor centers into the diamagnetic state Mox.     

The outer mitochondrial membrane protein mitoNEET contains a novel redox-active 2Fe-2S cluster

The outer mitochondrial membrane protein mitoNEET was discovered as a binding target of pioglitazone, an insulin-sensitizing drug of the thiazolidinedione class used to treat type 2 diabetes (Colca, J. R., McDonald, W. G., Waldon, D. J., Leone, J. W., Lull, J. M., Bannow, C. A., Lund, E. T., and Mathews, W. R. (2004) Am. J. Physiol. 286, E252-E260). We have shown that mitoNEET is a member of a small family of proteins containing a 39-amino-acid CDGSH domain. Although the CDGSH domain is annotated as a zinc finger motif, mitoNEET was shown to contain iron (Wiley, S. E., Murphy, A. N., Ross, S. A., van der Geer, P., and Dixon, J. E. (2007) Proc. Natl. Acad. Sci. U. S. A. 104, 5318-5323). Optical and electron paramagnetic resonance spectroscopy showed that it contained a redox-active pH-labile Fe-S cluster. Mass spectrometry showed the loss of 2Fe and 2S upon cofactor extrusion. Spectroscopic studies of recombinant proteins showed that the 2Fe-2S cluster was coordinated by Cys-3 and His-1. The His ligand was shown to be involved in the observed pH lability of the cluster, indicating that loss of this ligand via protonation triggered release of the cluster. mitoNEET is the first identified 2Fe-2S-containing protein located in the outer mitochondrial membrane. Based on the biophysical data and domain fusion analysis, mitoNEET may function in Fe-S cluster shuttling and/or in redox reactions.     

Metal use in ribonucleotide reductase R2, di-iron, di-manganese and heterodinuclear--an intricate bioinorganic workaround to use different metals for the same reaction

The ferritin-like superfamily comprises of several protein groups that utilize dinuclear metal sites for various functions, from iron storage to challenging oxidations of substrates. Ribonucleotide reductase R2 proteins use the metal site for the generation of a free radical required for the reduction of ribonucleotides to deoxyriboinucleotides, the building blocks of DNA. This ubiquitous and essential reaction has been studied for over four decades and the R2 proteins were, until recently, generally believed to employ the same cofactor and mechanism for radical generation. In this reaction, a stable tyrosyl radical is produced following activation and cleavage of molecular oxygen at a dinuclear iron site in the protein. Discoveries in the last few years have now firmly established that the radical generating reaction is not conserved among the R2 proteins but that different subgroups, that are structurally very similar, instead employ di-manganese or heterodinuclear Mn-Fe cofactors as radical generators. This is remarkable considering that the protein must exercise a strict control over oxygen activation, reactive metal-oxygen intermediate species and the resulting redox potential of the produced radical equivalent. Given the differences in redox properties between Mn and Fe, use of a different metal for this reaction requires associated adaptations of the R2 protein scaffold and the activation mechanism. Further analysis of the differences in protein sequence between R2 subgroups have also led to the discovery of new groups of R2-like proteins with completely different functions, expanding the chemical repertoire of the ferritin-like superfamily. This review describes the discoveries leading up to the identification of the different Mn-containing R2 protein groups and our current understanding of them. Hypotheses regarding the biochemical rationale to develop these chemically complex alternative solutions are also discussed.     

M?ssbauer study of Clostridium pasteurianum hydrogenase II. Evidence for a novel three-iron cluster

Hydrogenase II contains two iron-sulfur clusters, one of the [4Fe-4S] type and one of unknown structure with unusual spectral properties (H-cluster). Using M?ssbauer spectroscopy we have studied the H-cluster under a variety of conditions. In the reduced state the cluster exhibits, in zero magnetic field, spectra with the typical 2:1 quadrupole pattern of reduced [3Fe-4S] clusters. However, whereas the latter are paramagnetic (S = 2) the H-cluster is diamagnetic (S = 0). Upon oxidation and exposure to CO the H-cluster exhibits an S = 1/2 EPR spectrum with g values at 2.03, 2.02, and 2.00. In this state, the M?ssbauer spectra reveal two cluster subsites with magnetic hyperfine coupling constants AI = +26.5 MHz and AII = -30 MHz. ENDOR data obtained by Hoffman and co-workers (Telser, J., Benecky, M. J., Adams, M. W. W., Mortenson, L. E., and Hoffman, B. M. (1986) J. Biol. Chem. 261, 13536-13541) show a 57Fe resonance at AIII approximately equal to 9.5 MHz. Analysis of the M?ssbauer spectra shows that this resonance represents one iron site. Our studies of the reduced and CO-bound oxidized states of hydrogenase II suggest that the H-cluster contains three iron atoms. The data obtained for the oxidized H-cluster suggest a novel type of 3-Fe cluster and bear little resemblance to those reported for oxidized [3Fe-4S] clusters with g = 2.01 EPR signals. In the reduced sample the [4Fe-4S]1+ cluster appears to occur in a mixture of two distinct electronic states.     

Nitrogenase-catalyzed ethane production and CO-sensitive hydrogen evolution from MoFe proteins having amino acid substitutions in an alpha-subunit FeMo cofactor-binding domain.

Unlike wild type, certain Mo-dependent nitrogenases, which are expressed in non-N2-fixing mutant strains of Azotobacter vinelandii and have single amino acid substitutions within a region of the MoFe protein alpha-subunit proposed to encompass an FeMo cofactor-binding domain, are able to catalyze the reduction of acetylene by both two and four electrons to yield ethylene and ethane, respectively (Scott, D. J., May, H. D., Newton, W. E., Brigle, K. E., and Dean, D. R. (1990) Nature 343, 188-190). Although the V-dependent nitrogenase is also able to catalyze the reduction of acetylene to the same two- and four-electron products (Dilworth, M. J., Eady, R. R., Robson, R. L., and Miller, R. W. (1987) Nature 327, 167-168), we find that ethane formation from acetylene catalyzed by the altered Mo-dependent nitrogenases occurs by a different mechanism, which is distinguished by: (i) an increased sensitivity to CO; (ii) the absence of a lag; and (iii) no temperature dependence of product distribution among ethylene and ethane during acetylene reduction. An altered MoFe protein, which was purified from one such mutant strain having the alpha-subunit glutaminyl 191 residue substituted by lysyl, exhibited both a changed S = 3/2 EPR spectrum and changes in the distribution of electrons to various products when compared to wild type. Also, unlike wild type, this altered MoFe protein catalyzed proton reduction that is inhibited by carbon monoxide (CO). Because proton reduction catalyzed by a nitrogenase that has a FeMo cofactor with citrate rather than homocitrate as its organic constituent (Liang, J., Madden, M., Shah, V. K., and Burris, R. H. (1990) Biochemistry 29, 8577-8581) is also inhibited by CO, the possibility arose that changes in the polypeptide environment of FeMo cofactor might have caused a rearrangement in its molecular structure or composition. However, this possibility was ruled out by biochemical reconstitution studies (using FeMo cofactor isolated from both the wild-type and altered MoFe proteins), which were monitored by EPR spectroscopy and resulting catalytic activity.     

Growth and Nutrient Uptake by Barley (Hordeum vulgare L. cv Herta): Studies Using an N-(2-Hydroxyethyl)ethylenedinitrilotriacetic Acid-Buffered Nutrient Solution Technique (II. Role of Zinc in the Uptake and Root Leakage of Mineral Nutrients)

Barley seedlings (Hordeum vulgare L. cv Herta) were grown in N-(2-hydroxyethyl)ethylenedinitrilotriacetic acid-buffered nutrient solutions with or without adequate Zn supplies. Fifteen-d-old Zn-deficient seedlings contained higher concentrations of Mn, Ca, Mg, and P in their shoots and more Fe, Mn, Cu, K, Ca, and P in their roots than did similar Zn-adequate seedlings, confirming results reported in our companion study (W.A. Norvell and R.M. Welch [1993] Plant Physiol 101: 619-625). Zn-deficient roots leaked greater quantities of K, Mn, Cu, and Cl than did roots supplied adequately with Zn; they also leaked significant amounts of Zn even though the seedlings were not supplied Zn during growth. Calculated uptake rates of P, Mn, and Na were sharply reduced, but uptake rates of K and Mg were stimulated by increasing the Zn2+ activity in nutrient solutions. Intact roots of Zn-deficient seedlings contained lower concentrations of 5,5[prime] -dithio-bis(2-nitrobenzoic acid) reactive sulfhydryl groups in comparison to Zn-adequate roots. Apparently, Zn is required for the uptake and retention of several mineral nutrients by roots, possibly by playing a protective role in preventing the oxidation of sulfhydryl groups to disulfides in root-cell plasma membrane proteins involved in ion channel-gating phenomena.     

Nitrogenase X: M?ssbauer and EPR studies on reversibly oxidized MoFe protein from Azotobacter vinelandii OP. Nature of the iron centers.

Under anaerobic conditions the molybdenum-iron protein (MoFe protein) from Azotobacter vinelandii can be reversibly oxidized with thionine. Electron paramagnetic resonance studies reveal that the oxidation proceeds in two distinct phases: the MoFe protein can be oxidized by four electrons without loss of the EPR signal from the S = 3/2 cofactor centers. A second oxidation step, involving two electrons, leads to the disappearance of the cofactor EPR signal. In order to correlate the events during the thionine titration with redox reactions involving individual iron centers we have studied the MoFe proteins from A vinelandii and Clostridium pasteurianum with M?ssbauer spectroscopy. Spectra were taken in the temperature range from 1.5 K to 200 K in applied magnetic fields of up to 54 kG. Analysis of the M?ssbauer data allows us to draw three major conclusions: (1) the holoprotein contains 30 +/- 2 iron atoms. (2) Most probably, 12 iron atoms belong to two, apparently identical, iron clusters (labeled M) which we have shown previously to be structural components of the iron and molybdenum containing cofactor of nitrogenase. The M-centers can be stabilized in three distinct oxidation states, MOXe- in equilibrium MNe- in equilibrium MR. The diamagnetic (S = 0) state MOX is attained by oxidation of the native state MN with either thionine or oxygen. MR is observed under nitrogen fixing conditions. (3) The data strongly suggest that 16 iron atoms are associated with four iron centers which we propose to call P-clusters. Each P-cluster contains four spin-coupled iron atoms. In the native protein the P-clusters are in the diamagnetic state PN, yielding the M?ssbauer signature which we have labeled previously 'components D and Fe2+'. Three irons of the D-type and one iron of the Fe2+-type appear to comprise a P-cluster. A one-electron oxidation yields the paramagnetic state POX. Although the state POX is characterized by half-integral electronic spin a peculiar combination of zero-field splitting parameters and spin relaxation renders this state EPR-silent. Spectroscopically, the P-clusters are novel structures; there is, however, evidence that they are closely related to familiar 4Fe-4S centers.     

Inactivation of Escherichia coli glutamine synthetase by xanthine oxidase, nicotinate hydroxylase, horseradish peroxidase, or glucose oxidase: effects of ferredoxin, putidaredoxin, and menadione

Previous studies have shown that several mixed-function oxidation (MFO) systems are capable of catalyzing the inactivation of glutamine synthetase (GS) [R.L. Levine, C. N. Oliver, R. M. Fulks, and E. R. Stadtman (1978) Proc. Natl. Acad. Sci. USA 78, 2120-2124] and a number of the other enzymes [L. Fucci, C. N. Oliver, M. J. Coon, and E. R. Stadtman (1983) Proc. Natl. Acad. Sci. USA 80, 1521-1525]. It has now been found that in the presence of Fe(III), O2, and an appropriate electron donor (hypoxanthine or NADPH, respectively) glutamine synthetase is also inactivated by either milk xanthine oxidase or Clostridial nicotinate hydroxylase. Inactivation of glutamine synthetase by either of these flavoproteins is greatly stimulated by the presence of electron carrier proteins possessing nonheme-iron-sulfur (NHIS) clusters (i.e., ferredoxin or putidaredoxin) or by the presence of menadione. The inactivation reactions are partially inhibited by free radical scavengers, superoxide dismutase, (SOD), histidine, mannitol, dimethyl sulfoxide, and dimethylthiourea, and are inhibited completely by either Mn(II), EDTA, or catalase. The sensitivity to SOD inhibition is greatly suppressed when the xanthine oxidase system is supplemented with either ferredoxin or redoxin. In the presence of the latter NHIS-proteins (and only when they are present), MFO systems, comprised of either horseradish peroxidase and H2O2 or glucose oxidase, O2, and glucose, can also catalyze the inactivation of GS. The ability of ferredoxin and putidaredoxin to promote oxidation modification of GS by any one of these MFO systems suggests that proteins with NHIS centers may mediate the generation (or stabilization) of highly reactive radical intermediates.     

Paramagnetic ion effects on the nuclear magnetic resonance spectrum of transfer ribonucleic acid: assignment of the 15--48 tertiary resonance.

We have studied the effects of Co2+ and Mn2+ ions on the low-field nuclear magnetic resonance (NMR) spectra of pure class 1 transfer ribonucleic acid (tRNA) species. With 1.2 mM tRNA in the presence of 15 mM MgCl2 discrete paramagnetic effects were observed for Co2+ at concentrations in the range 0.02--0.1 mM and for Mn2+ in the range 0.002--0.01 mM, indicating fast exchange of these cations with tRNA. Both of these cations paramagnetically relax the s4U8--A14 resonance as well as other resonances from proximal base pairs. The Co2+ site appears to be the same site on G15 which was observed crystallographically [Jack, A., Ladner, J. E., Rhodes, D., Brown, R. S., & Klug, A. (1977) J. Mol. Biol. 111, 315-328]; the initially occupied tight Mn2+ site is the cation site involving the phosphate of U8. There are three base pairs within 10 A of both sites, namely, G15--C48, A14--s4U8, and C13--G22; this has led to the assignment of the G15--C48 and C13--G22 resonances in the NMR spectrum [Jack, A., Ladner, J. E., Rhodes, D., Brown, R. S., & Klug, A. (1977) J. Mol. Biol. 111, 315--328; Holbrook, S. R., Sussman, J. L., Warrant, R. W., Church, G. M., & Kim, Sung-Hou (1977) Nucleic Acids Res. 4, 2811--2820; Quigley, G. J., Teeter, M. M., & Rich, A. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 64--68].