The Experts below are selected from a list of 900 Experts worldwide ranked by ideXlab platform

M. Elizabeth Stroupe - One of the best experts on this subject based on the ideXlab platform.

  • The Siroheme-[4Fe-4S] Coupled Center.
    Metal ions in life sciences, 2020
    Co-Authors: Isabel Askenasy, M. Elizabeth Stroupe
    Abstract:

    In nature, sulfur exists in a range of oxidation states and the two-electron reduced form is the most commonly found in biomolecules like the sulfur-containing amino acids cysteine and methionine, some cofactors, and polysaccharides. Sulfur is reduced through two pathways: dissimilation, where sulfite (SO2-3) is used as terminal electron acceptor; and assimilation, where sulfite is reduced to sulfide (S2-) for incorporation into biomass. The pathways are independent, but share the sulfite reductase function, in which a single enzyme reduces sulfite by six electrons to make sulfide. With few exceptions, sulfite reductases from either pathway are iron metalloenzymes with structurally diverse configurations that range from monomers to tetramers. The hallmark of sulfite reductase is its catalytic center made of an iron-containing porphyrinoid called Siroheme that is covalently coupled to a [4Fe-4S] cluster through a shared cysteine ligand. The substrate evolves through a push-pull mechanism, where electron transfer is coupled to three dehydration steps. Siroheme is an isobacteriochlorin that is more readily oxidized than protoporphyin IX-derived hemes. It is synthesized from uroporphyrinogen III in three steps (methylation, a dehydrogenation, and ferrochelation) that are performed by enzymes with homology to those involved in cobalamin synthesis. Future research will need to address how the Siroheme-[4Fe-4S] clusters are assembled into apo-sulfite and nitrite reductases. The chapter will discuss how environmental microbes use sulfite reductase to survive in a range of ecosystems; how atomic-resolution structures of dissimilatory and assimilatory sulfite reductases reveal their ancient homology; how the Siroheme-[4Fe-4S] cluster active site catalyzes the six-electron reduction of sulfite to sulfide; and how Siroheme is synthesized across diverse microrganisms.

  • Siroheme synthase orients substrates for dehydrogenase and chelatase activities in a common active site
    Nature Communications, 2020
    Co-Authors: Joseph M Pennington, Lauren Mcgarry, M Kemp, Yu Chen, M. Elizabeth Stroupe
    Abstract:

    Siroheme is the central cofactor in a conserved class of sulfite and nitrite reductases that catalyze the six-electron reduction of sulfite to sulfide and nitrite to ammonia. In Salmonella enterica serovar Typhimurium, Siroheme is produced by a trifunctional enzyme, Siroheme synthase (CysG). A bifunctional active site that is distinct from its methyltransferase activity catalyzes the final two steps, NAD+-dependent dehydrogenation and iron chelation. How this active site performs such different chemistries is unknown. Here, we report the structures of CysG bound to precorrin-2, the initial substrate; sirohydrochlorin, the dehydrogenation product/chelation substrate; and a cobalt-sirohydrochlorin product. We identified binding poses for all three tetrapyrroles and tested the roles of specific amino acids in both activities to give insights into how a bifunctional active site catalyzes two different chemistries and acts as an iron-specific chelatase in the final step of Siroheme synthesis. Siroheme is an essential bacterial iron tetrapyrrole used by Siroheme-dependent sulfite and nitrite reductases. Here the authors shed light on the catalytic mechanisms of Siroheme synthase through the structures of the bifunctional dehydrogenase/chelatase CysG module bound to its substrate, precorrin-2, the product/substrate sirohydrochlorin, and cobalt-sirohydrochlorin.

  • The role of extended Fe4S4cluster ligands in mediating sulfite reductase hemoprotein activity.
    Biochimica et Biophysica Acta, 2018
    Co-Authors: Marisa R. Cepeda, Joseph M Pennington, Lauren Mcgarry, J Krzystek, M. Elizabeth Stroupe
    Abstract:

    Abstract The Siroheme-containing subunit from the multimeric hemoflavoprotein NADPH-dependent sulfite reductase (SiR/SiRHP) catalyzes the six electron-reduction of SO32− to S2−. Siroheme is an iron-containing isobacteriochlorin that is found in sulfite and homologous Siroheme-containing nitrite reductases. Siroheme does not work alone but is covalently coupled to a Fe4S4 cluster through one of the cluster's ligands. One long-standing hypothesis predicted from this observation is that the environment of one iron-containing cofactor influences the properties of the other. We tested this hypothesis by identifying three amino acids (F437, M444, and T477) that interact with the Fe4S4 cluster and probing the effect of altering them to alanine on the function and structure of the resulting enzymes by use of activity assays, X-ray crystallographic analysis, and EPR spectroscopy. We showed that F437 and M444 gate access for electron transfer to the Siroheme-cluster assembly and the direct hydrogen bond between T477 and one of the cluster sulfides is important for determining the geometry of the Siroheme active site.

  • The role of extended Fe4S4 cluster ligands in mediating sulfite reductase hemoprotein activity.
    Biochimica et biophysica acta. Proteins and proteomics, 2018
    Co-Authors: Marisa R. Cepeda, Joseph M Pennington, Lauren Mcgarry, J Krzystek, M. Elizabeth Stroupe
    Abstract:

    The Siroheme-containing subunit from the multimeric hemoflavoprotein NADPH-dependent sulfite reductase (SiR/SiRHP) catalyzes the six electron-reduction of SO32- to S2-. Siroheme is an iron-containing isobacteriochlorin that is found in sulfite and homologous Siroheme-containing nitrite reductases. Siroheme does not work alone but is covalently coupled to a Fe4S4 cluster through one of the cluster's ligands. One long-standing hypothesis predicted from this observation is that the environment of one iron-containing cofactor influences the properties of the other. We tested this hypothesis by identifying three amino acids (F437, M444, and T477) that interact with the Fe4S4 cluster and probing the effect of altering them to alanine on the function and structure of the resulting enzymes by use of activity assays, X-ray crystallographic analysis, and EPR spectroscopy. We showed that F437 and M444 gate access for electron transfer to the Siroheme-cluster assembly and the direct hydrogen bond between T477 and one of the cluster sulfides is important for determining the geometry of the Siroheme active site.

  • Mutational analysis of sulfite reductase hemoprotein reveals the mechanism for coordinated electron and proton transfer.
    Biochemistry, 2012
    Co-Authors: K.w. Smith, M. Elizabeth Stroupe
    Abstract:

    Sulfite reductase catalyzes the six-electron reduction of sulfite to sulfide. The active site, found in the hemoprotein subunit (SiRHP), sits on the distal face of a negatively charged porphyrinoid called Siroheme whose central iron atom is coupled to a proximal Fe4S4 cluster. Four positively charged amino acids are positioned around the active site cavity. Together, these two arginines (R83 and R153) and two lysines (K215 and K217) mitigate the negative charge on the Siroheme macrocycle. They also serve as a cage around the distally bound anion that tightens when substrate binds and an active site loop clamps down. Structures of native SiRHP point to these amino acids as being important, but their specific roles are ill-defined. Here, we have altered those four active site amino acids and one amino acid on the flexible loop (N149) to probe their roles in SiRHP activity. None of these positively charged residues is required for electron transfer, but only R83S and N149W variants can produce a fully reduce...

David B. Knaff - One of the best experts on this subject based on the ideXlab platform.

  • New Insights into the Catalytic Cycle of Plant Nitrite Reductase. Electron Transfer Kinetics and Charge Storage
    Biochemistry, 2009
    Co-Authors: Pierre Sétif, Nicolas Cassan, Bernard Lagoutte, Masakazu Hirasawa, Jatindra N Tripathy, David B. Knaff
    Abstract:

    Nitrite reductase, which reduces nitrite to ammonium in a six-electron reaction, was characterized through kinetic analysis of an electron transfer cascade involving photoexcited Photosystem I and ferredoxin. This cascade was studied at physiological pH by flash-absorption spectroscopy. Two different forms of the enzyme were studied: one isolated from spinach leaf and one histidine-tagged recombinant form. When the enzyme is oxidized in the absence of nitrite, single-enzyme reduction leads mostly to Siroheme reduction with the leaf enzyme, whereas the Siroheme and the [4Fe-4S] cluster are both reduced in equivalent amounts in the recombinant enzyme. When combined with the results of deazaflavin/EDTA photoreduction experiments, these data support a 50 mV negative shift of the Siroheme midpoint potential in the recombinant enzyme. Despite this difference, the two forms of the enzyme exhibit similar values for the rate constant of single reduction by reduced ferredoxin (1200 s−1) and for kcat (420−450 electr...

  • structure of spinach nitrite reductase implications for multi electron reactions by the iron sulfur Siroheme cofactor
    Biochemistry, 2005
    Co-Authors: Uma Swamy, Masakazu Hirasawa, David B. Knaff, Jatindra N Tripathy, Meitian Wang, James P Allen
    Abstract:

    The structure of nitrite reductase, a key enzyme in the process of nitrogen assimilation, has been determined using X-ray diffraction to a resolution limit of 2.8 A. The protein has a globular fold consisting of 3 α/β domains with the Siroheme−iron sulfur cofactor at the interface of the three domains. The Fe4S4 cluster is coordinated by cysteines 441, 447, 482, and 486. The Siroheme is located at a distance of 4.2 A from the cluster, and the central iron atom is coordinated to Cys 486. The Siroheme is surrounded by several ionizable amino acid residues that facilitate the binding and subsequent reduction of nitrite. A model for the ferredoxin:nitrite reductase complex is proposed in which the binding of ferredoxin to a positively charged region of nitrite reductase results in elimination of exposure of the cofactors to the solvent. The structure of nitrite reductase shows a broad similarity to the hemoprotein subunit of sulfite reductase but has many significant differences in the backbone positions that...

  • Structure of Spinach Nitrite Reductase: Implications for Multi-electron Reactions by the Iron-Sulfur:Siroheme Cofactor
    Biochemistry, 2005
    Co-Authors: Uma Swamy, Masakazu Hirasawa, David B. Knaff, Jatindra N Tripathy, Meitian Wang, Sung Kun Kim, James P Allen
    Abstract:

    The structure of nitrite reductase, a key enzyme in the process of nitrogen assimilation, has been determined using X-ray diffraction to a resolution limit of 2.8 A. The protein has a globular fold consisting of 3 alpha/beta domains with the Siroheme-iron sulfur cofactor at the interface of the three domains. The Fe(4)S(4) cluster is coordinated by cysteines 441, 447, 482, and 486. The Siroheme is located at a distance of 4.2 A from the cluster, and the central iron atom is coordinated to Cys 486. The Siroheme is surrounded by several ionizable amino acid residues that facilitate the binding and subsequent reduction of nitrite. A model for the ferredoxin:nitrite reductase complex is proposed in which the binding of ferredoxin to a positively charged region of nitrite reductase results in elimination of exposure of the cofactors to the solvent. The structure of nitrite reductase shows a broad similarity to the hemoprotein subunit of sulfite reductase but has many significant differences in the backbone positions that could reflect sequence differences or could arise from alterations of the sulfite reductase structure that arise from the isolation of this subunit from the native complex. The implications of the nitrite reductase structure for understanding multi-electron processes are discussed in terms of differences in the protein environments of the cofactors.

  • Reactions of spinach nitrite reductase with its substrate, nitrite, and a putative intermediate, hydroxylamine.
    Biochemistry, 2004
    Co-Authors: Sofya Kuznetsova, Pierre Sétif, Masakazu Hirasawa, David B. Knaff, Tony A Mattioli
    Abstract:

    Plant nitrite reductase (NiR) catalyzes the reduction of nitrite (NO2-) to ammonia, using reduced ferredoxin as the electron donor. NiR contains a [4Fe-4S] cluster and an Fe-Siroheme, which is the nitrite binding site. In the enzyme's as-isolated form ([4Fe-4S]2+/Fe3+), resonance Raman spectroscopy indicated that the Siroheme is in the high-spin ferric hexacoordinated state with a weak sixth axial ligand. Kinetic and spectroscopic experiments showed that the reaction of NiR with NO2- results in an unexpectedly EPR-silent complex formed in a single step with a rate constant of 0.45 ± 0.01 s-1. This binding rate is slow compared to that expected from the NiR turnover rates reported in the literature, suggesting that binding of NO2- to the as-isolated form of NiR is not the predominant type of substrate binding during enzyme turnover. Resonance Raman spectroscopic characterization of this complex indicated that (i) the Siroheme iron is low-spin hexacoordinated ferric, (ii) the ligand coordination is unusuall...

  • Oxidation-reduction properties of maize ferredoxin: sulfite oxidoreductase.
    Biochimica et biophysica acta, 2004
    Co-Authors: Masakazu Hirasawa, Toshiharu Hase, Masato Nakayama, David B. Knaff
    Abstract:

    Oxidation-reduction titrations have been carried out on the wild-type, ferredoxin-dependent sulfite reductase from maize and two site-specific variants of the enzyme. E(m) values have been determined for the Siroheme and [4Fe-4S] cluster prosthetic groups of the enzyme, which titrate as independent, one-electron carriers. Visible-region difference spectra suggest that reduction of the [4Fe-4S] cluster significantly perturbs the spectrum of the reduced Siroheme group of the enzyme. The effects of Siroheme axial ligation, by either cyanide or phosphate ligands, on the redox properties of sulfite reductase have also been examined. For comparison, the effects of phosphate and cyanide on the redox properties of the ferredoxin-dependent nitrite reductase of spinach chloroplasts, an enzyme with the same prosthetic group arrangement as sulfite reductase, have been examined.

Wilfred R. Hagen - One of the best experts on this subject based on the ideXlab platform.

  • The dissimilatory sulfite reductase from Desulfosarcina variabilis is a desulforubidin containing uncoupled metalated Sirohemes and S = 9/2 iron-sulfur clusters.
    Biochemistry, 1993
    Co-Authors: Alexander F. Arendsen, Ronnie B. G. Wolbert, Marc F J M Verhagen, Antonio J Pierik, Alfons J M Stams, Mike S. M. Jetten, Wilfred R. Hagen
    Abstract:

    The active site of Escherichia coli NADPH-sulfite reductase has previously been modeled as a Siroheme with its iron bridged to a nearby iron-sulfur cubane, resulting in antiferromagnetic exchange coupling between all iron atoms. The model has been suggested to hold also for other sulfite reductases and nitrite reductases. We have recently challenged the generality of the model with the finding that the EPR of Fe/S in dissimilatory sulfite reductase (desulfoviridin) from Desulfovibrio vulgaris indicates that an S = 9/2 system is not subject to coupling. Siroheme in desulfoviridin is to a large extent demetalated, and therefore coupling is physically impossible. We have now studied examples from a second class of dissimilatory sulfite reductases, desulforubidins, which have their siroporphyrins fully metalated. Desulforubidin from Desulfosarcina variabilis is a 208-kDa alpha 2 beta 2 gamma 2 hexamer. The alpha- and beta-subunits are immunologically active with antibodies raised against the corresponding subunits from D. vulgaris desulfoviridin, whereas the gamma-subunit is not. The desulforubidin contains two fully metalated Sirohemes and a total of approximately 15 Fe and approximately 19 S2-. Quantification of high-spin plus low-spin heme EPR signals accounts for all sirohydrochlorin. The frequency-independent (9-35 GHz) effective perpendicular g-values of the high-spin S = 5/2 Siroheme (6.33, 5.19) point to quantum mixing with an excited (approximately 770 cm-1) S = 3/2 multiplet. Similar anomalous g-values are observed with sulfite reductases from Desulfovibrio baarsii and Desulfotomaculum acetoxidans. The D. variabilis enzyme exhibits very approximately stoichiometric S = 9/2 EPR (g = 16).(ABSTRACT TRUNCATED AT 250 WORDS)

  • The dissimilatory sulfite reductase from Desulfosarcina variabilis is a desulforubidin containing uncoupled metalated Sirohemes and S = 9/2 iron-sulfur clusters
    Biochemistry, 1993
    Co-Authors: Alexander F. Arendsen, Ronnie B. G. Wolbert, Marc F J M Verhagen, Antonio J Pierik, Alfons J M Stams, Mike S. M. Jetten, Wilfred R. Hagen
    Abstract:

    The active site of Escherichia coli NADPH-sulfite reductase has previously been modeled as a Siroheme with its iron bridged to a nearby iron-sulfur cubane, resulting in antiferromagnetic exchange coupling between all iron atoms. The model has been suggested to hold also for other sulfite reductases and nitrite reductases. We have recently challenged the generality of the model with the finding that the EPR of Fe/S in dissimilatory sulfite reductase (desulfoviridin) from Desulfovibrio vulgaris indicates that an S=9/2 system is not subject to coupling. Siroheme in desulfoviridin is to a large extent demetalated, and therefore coupling is physically impossible. We have now studied examples from a second class of dissimilatory sulfite reductases, desulforubidins, which have their siroporphyrins fully metalated

  • S= 9/2 EPR signals are evidence against coupling between the Siroheme and the Fe/S cluster prosthetic groups in Desulfovibrio vulgaris (Hildenborough) dissimilatory sulfite reductase
    European journal of biochemistry, 1991
    Co-Authors: Antonio J Pierik, Wilfred R. Hagen
    Abstract:

    Sulfite reductases contain Siroheme and iron-sulfur cluster prosthetic groups. The two groups are believed to be structurally linked via a single, common ligand. This chemical model is based on a magnetic model for the oxidized enzyme in which all participating iron ions are exchange coupled. This description leads to two serious discrepancies. Although the iron-sulfur cluster is assumed to be a diamagnetic cubane, [4Fe-4S]2+, all iron appears to be paramagnetic in Mossbauer spectroscopy. On the other hand, EPR spectroscopy has failed to detect anything but a single high-spin heme. We have re-addressed this problem by searching for new EPR spectroscopic clues in concentrated samples of dissimilatory sulfite reductase from Desulfovibrio vulgaris (Hildenborough). We have found several novel signals with effective g values of 17, 15.1, 11.7, 9.4, 9.0, 4. The signals are interpreted in terms of an S = 9/2 system with spin-Hamiltonian parameters g = 2.00, D = -0.56 cm-1, magnitude of E/D = 0.13 for the major component. In a reductive titration with sodium borohydride the spectrum disappears with Em = -205 mV at pH 7.5. Contrarily, the major high-spin Siroheme component has S = 5/2, g = 1.99, D = +9 cm-1, magnitude of E/D = 0.042, and Em = -295 mV. The sum of all Siroheme signals integrates to 0.2 spin/half molecule, indicating considerable demetallation of this prosthetic group. Rigorous quantification procedures for S = 9/2 are not available, however, estimation by an approximate method indicates 0.6 S = 9/2 spin/half molecule. The S = 9/2 system is ascribed to an iron-sulfur cluster. It follows that this cluster is probably not a cubane, is not necessarily exchange-coupled to the Siroheme, and, therefore, is not necessarily structurally close to the Siroheme. It is suggested that this iron-sulfur prosthetic group has a novel structure suitable for functioning in multiple electron transfer.

  • s 9 2 epr signals are evidence against coupling between the Siroheme and the fe s cluster prosthetic groups in desulfovibrio vulgaris hildenborough dissimilatory sulfite reductase
    FEBS Journal, 1991
    Co-Authors: Antonio J Pierik, Wilfred R. Hagen
    Abstract:

    Sulfite reductases contain Siroheme and iron-sulfur cluster prosthetic groups. The two groups are believed to be structurally linked via a single, common ligand. This chemical model is based on a magnetic model for the oxidized enzyme in which all participating iron ions are exchange coupled. This description leads to two serious discrepancies. Although the iron-sulfur cluster is assumed to be a diamagnetic cubane, [4Fe–4S]2+, all iron appears to be paramagnetic in Mossbauer spectroscopy. On the other hand, EPR spectroscopy has failed to detect anything but a single high-spin heme. We have re-addressed this problem by searching for new EPR spectroscopic clues in concentrated samples of dissimilatory sulfite reductase from Desulfovibrio vulgaris (Hildenborough). We have found several novel signals with effective g values of 17, 15.1, 11.7, 9.4, 9.0, 4. The signals are interpreted in terms of an S= 9/2 system with spin-Hamiltonian parameters g= 2.00, D=−0.56 cm−1, |E/D|= 0.13 for the major component. In a reductive titration with sodium borohydride the spectrum disappears with Em=−205 mV at pH 7.5. Contrarily, the major high-spin Siroheme component has S= 5/2, g= 1.99, D=+9 cm−1, |E/D|= 0.042, and Em=−295 mV. The sum of all Siroheme signals integrates to 0.2 spin/half molecule, indicating considerable demetallation of this prosthetic group. Rigorous quantification procedures for S= 9/2 are not available, however, estimation by an approximate method indicates 0.6 S= 9/2 spin/half molecule. The S= 9/2 system is ascribed to an iron-sulfur cluster. It follows that this cluster is probably not a cubane, is not necessarily exchange-coupled to the Siroheme, and, therefore, is not necessarily structurally close to the Siroheme. It is suggested that this iron-sulfur prosthetic group has a novel structure suitable for functioning in multiple electron transfer.

Martin J. Warren - One of the best experts on this subject based on the ideXlab platform.

  • 2014b) Identification and characterization of the ‘missing’ terminal enzyme for Siroheme biosynthesis in alpha-proteobacteria. Mol Microbiol 92
    2016
    Co-Authors: Shilpa Bali, Sarah E Rollauer, Evelyne Raux-deery, Pietro Roversi, Martin J. Warren, Susan M. Lea, Stuart J Ferguson
    Abstract:

    It has recently been shown that the biosynthetic route for both the d1-haem cofactor of dissimilatory cd1 nitrite reductases and haem, via the novel alternative-haem-synthesis pathway, involves Siroheme as an intermediate, which was previously thought to occur only as a cofactor in assimilatory sulphite/ nitrite reductases. In many denitrifiers (which require d1-haem), the pathway to make Siroheme remained to be identified. Here we identify and characterize a sirohydrochlorin–ferrochelatase from Paracoccus pantotrophus that catalyses the last step of Siroheme synthesis. It is encoded by a gene annotated as cbiX that was previously assumed to be encoding a cobal-tochelatase, acting on sirohydrochlorin. Expressing this chelatase from a plasmid restored the wild-type phenotype of an Escherichia coli mutant-strain lacking sirohydrochlorin–ferrochelatase activity, showing that this chelatase can act in the in vivo Siroheme synthesis. A ΔcbiX mutant in P. denitrificans was unable to respire anaerobically on nitrate, proving the role of Siroheme as a precursor to another cofactor. We report the 1.9 Å crystal structure of this ferrochelatase. In vivo analysis of single amino acid variants of this chelatase suggests that two histidines, His127 and His187, are essential for Siroheme synthesis. This CbiX can generally be identified in α-proteobacteria as the terminal enzyme of Siroheme biosynthesis

  • identification and characterization of the missing terminal enzyme for Siroheme biosynthesis in α proteobacteria
    Molecular Microbiology, 2014
    Co-Authors: Shilpa Bali, Sarah E Rollauer, Evelyne Rauxdeery, Pietro Roversi, Martin J. Warren, Stuart J Ferguson
    Abstract:

    It has recently been shown that the biosynthetic route for both the d1-haem cofactor of dissimilatory cd1 nitrite reductases and haem, via the novel alternative-haem-synthesis pathway, involves Siroheme as an intermediate, which was previously thought to occur only as a cofactor in assimilatory sulphite/nitrite reductases. In many denitrifiers (which require d1-haem), the pathway to make Siroheme remained to be identified. Here we identify and characterize a sirohydrochlorin–ferrochelatase from Paracoccus pantotrophus that catalyses the last step of Siroheme synthesis. It is encoded by a gene annotated as cbiX that was previously assumed to be encoding a cobaltochelatase, acting on sirohydrochlorin. Expressing this chelatase from a plasmid restored the wild-type phenotype of an Escherichia coli mutant-strain lacking sirohydrochlorin–ferrochelatase activity, showing that this chelatase can act in the in vivo Siroheme synthesis. A ΔcbiX mutant in P. denitrificans was unable to respire anaerobically on nitrate, proving the role of Siroheme as a precursor to another cofactor. We report the 1.9 A crystal structure of this ferrochelatase. In vivo analysis of single amino acid variants of this chelatase suggests that two histidines, His127 and His187, are essential for Siroheme synthesis. This CbiX can generally be identified in α-proteobacteria as the terminal enzyme of Siroheme biosynthesis.

  • Identification and characterization of the ‘missing’ terminal enzyme for Siroheme biosynthesis in α‐proteobacteria
    Molecular Microbiology, 2014
    Co-Authors: Shilpa Bali, Sarah E Rollauer, Evelyne Raux-deery, Pietro Roversi, Martin J. Warren, Stuart J Ferguson
    Abstract:

    It has recently been shown that the biosynthetic route for both the d1-haem cofactor of dissimilatory cd1 nitrite reductases and haem, via the novel alternative-haem-synthesis pathway, involves Siroheme as an intermediate, which was previously thought to occur only as a cofactor in assimilatory sulphite/nitrite reductases. In many denitrifiers (which require d1-haem), the pathway to make Siroheme remained to be identified. Here we identify and characterize a sirohydrochlorin–ferrochelatase from Paracoccus pantotrophus that catalyses the last step of Siroheme synthesis. It is encoded by a gene annotated as cbiX that was previously assumed to be encoding a cobaltochelatase, acting on sirohydrochlorin. Expressing this chelatase from a plasmid restored the wild-type phenotype of an Escherichia coli mutant-strain lacking sirohydrochlorin–ferrochelatase activity, showing that this chelatase can act in the in vivo Siroheme synthesis. A ΔcbiX mutant in P. denitrificans was unable to respire anaerobically on nitrate, proving the role of Siroheme as a precursor to another cofactor. We report the 1.9 A crystal structure of this ferrochelatase. In vivo analysis of single amino acid variants of this chelatase suggests that two histidines, His127 and His187, are essential for Siroheme synthesis. This CbiX can generally be identified in α-proteobacteria as the terminal enzyme of Siroheme biosynthesis.

  • molecular hijacking of Siroheme for the synthesis of heme and d1 heme
    Proceedings of the National Academy of Sciences of the United States of America, 2011
    Co-Authors: Shilpa Bali, Susana A L Lobo, BERNARD THOMAS GOLDING, Mark J Howard, Ligia M Saraiva, David Palmer, A Lawrence, Stuart J Ferguson, Martin J. Warren
    Abstract:

    Modified tetrapyrroles such as chlorophyll, heme, Siroheme, vitamin B12, coenzyme F430, and heme d1 underpin a wide range of essential biological functions in all domains of life, and it is therefore surprising that the syntheses of many of these life pigments remain poorly understood. It is known that the construction of the central molecular framework of modified tetrapyrroles is mediated via a common, core pathway. Herein a further branch of the modified tetrapyrrole biosynthesis pathway is described in denitrifying and sulfate-reducing bacteria as well as the Archaea. This process entails the hijacking of Siroheme, the prosthetic group of sulfite and nitrite reductase, and its processing into heme and d1 heme. The initial step in these transformations involves the decarboxylation of Siroheme to give didecarboxySiroheme. For d1 heme synthesis this intermediate has to undergo the replacement of two propionate side chains with oxygen functionalities and the introduction of a double bond into a further peripheral side chain. For heme synthesis didecarboxySiroheme is converted into Fe-coproporphyrin by oxidative loss of two acetic acid side chains. Fe-coproporphyrin is then transformed into heme by the oxidative decarboxylation of two propionate side chains. The mechanisms of these reactions are discussed and the evolutionary significance of another role for Siroheme is examined.

  • Structure and function of SirC from Bacillus megaterium - a metal binding precorrin-2 dehydrogenase
    Biochemical Journal, 2008
    Co-Authors: Heidi L Schubert, Helen K Leech, Ruth S Rose, Amanda A Brindley, Christopher P Hill, Stephen E. J. Rigby, Martin J. Warren
    Abstract:

    In Bacillus megaterium, the synthesis of vitamin B12 (cobalamin) and Siroheme diverge at sirohydrochlorin along the branched modified tetrapyrrole biosynthetic pathway. This key intermediate is made by the action of SirC, a precorrin-2 dehydrogenase that requires NAD+ as a cofactor. The structure of SirC has now been solved by X-ray crystallography to 2.8 Å resolution. The protein is shown to consist of three domains and has a similar topology to the multifunctional Siroheme synthases Met8p and the N-terminal region of CysG, both of which catalyse not only the dehydrogenation of precorrin-2 but also the ferrochelation of sirohydrochlorin to give Siroheme. Guided by the structure, a number of active site residues within SirC were investigated by site-directed mutagenesis. No active site general base was identified, although surprisingly some of the resulting protein variants were found to have significantly enhanced catalytic activity. Unexpectedly, SirC was found to bind metal ions such as cobalt and copper, and to bind them in an identical fashion to that observed in Met8p. It is suggested that SirC may have evolved from a Met8p-like protein by loss of its chelatase activity. It is proposed that the ability of SirC to act as a single monofunctional enzyme, in conjunction with an independent chelatase, may provide greater control over the intermediate at this branchpoint in the synthesis of Siroheme and cobalamin.

Masakazu Hirasawa - One of the best experts on this subject based on the ideXlab platform.

  • New Insights into the Catalytic Cycle of Plant Nitrite Reductase. Electron Transfer Kinetics and Charge Storage
    Biochemistry, 2009
    Co-Authors: Pierre Sétif, Nicolas Cassan, Bernard Lagoutte, Masakazu Hirasawa, Jatindra N Tripathy, David B. Knaff
    Abstract:

    Nitrite reductase, which reduces nitrite to ammonium in a six-electron reaction, was characterized through kinetic analysis of an electron transfer cascade involving photoexcited Photosystem I and ferredoxin. This cascade was studied at physiological pH by flash-absorption spectroscopy. Two different forms of the enzyme were studied: one isolated from spinach leaf and one histidine-tagged recombinant form. When the enzyme is oxidized in the absence of nitrite, single-enzyme reduction leads mostly to Siroheme reduction with the leaf enzyme, whereas the Siroheme and the [4Fe-4S] cluster are both reduced in equivalent amounts in the recombinant enzyme. When combined with the results of deazaflavin/EDTA photoreduction experiments, these data support a 50 mV negative shift of the Siroheme midpoint potential in the recombinant enzyme. Despite this difference, the two forms of the enzyme exhibit similar values for the rate constant of single reduction by reduced ferredoxin (1200 s−1) and for kcat (420−450 electr...

  • structure of spinach nitrite reductase implications for multi electron reactions by the iron sulfur Siroheme cofactor
    Biochemistry, 2005
    Co-Authors: Uma Swamy, Masakazu Hirasawa, David B. Knaff, Jatindra N Tripathy, Meitian Wang, James P Allen
    Abstract:

    The structure of nitrite reductase, a key enzyme in the process of nitrogen assimilation, has been determined using X-ray diffraction to a resolution limit of 2.8 A. The protein has a globular fold consisting of 3 α/β domains with the Siroheme−iron sulfur cofactor at the interface of the three domains. The Fe4S4 cluster is coordinated by cysteines 441, 447, 482, and 486. The Siroheme is located at a distance of 4.2 A from the cluster, and the central iron atom is coordinated to Cys 486. The Siroheme is surrounded by several ionizable amino acid residues that facilitate the binding and subsequent reduction of nitrite. A model for the ferredoxin:nitrite reductase complex is proposed in which the binding of ferredoxin to a positively charged region of nitrite reductase results in elimination of exposure of the cofactors to the solvent. The structure of nitrite reductase shows a broad similarity to the hemoprotein subunit of sulfite reductase but has many significant differences in the backbone positions that...

  • Structure of Spinach Nitrite Reductase: Implications for Multi-electron Reactions by the Iron-Sulfur:Siroheme Cofactor
    Biochemistry, 2005
    Co-Authors: Uma Swamy, Masakazu Hirasawa, David B. Knaff, Jatindra N Tripathy, Meitian Wang, Sung Kun Kim, James P Allen
    Abstract:

    The structure of nitrite reductase, a key enzyme in the process of nitrogen assimilation, has been determined using X-ray diffraction to a resolution limit of 2.8 A. The protein has a globular fold consisting of 3 alpha/beta domains with the Siroheme-iron sulfur cofactor at the interface of the three domains. The Fe(4)S(4) cluster is coordinated by cysteines 441, 447, 482, and 486. The Siroheme is located at a distance of 4.2 A from the cluster, and the central iron atom is coordinated to Cys 486. The Siroheme is surrounded by several ionizable amino acid residues that facilitate the binding and subsequent reduction of nitrite. A model for the ferredoxin:nitrite reductase complex is proposed in which the binding of ferredoxin to a positively charged region of nitrite reductase results in elimination of exposure of the cofactors to the solvent. The structure of nitrite reductase shows a broad similarity to the hemoprotein subunit of sulfite reductase but has many significant differences in the backbone positions that could reflect sequence differences or could arise from alterations of the sulfite reductase structure that arise from the isolation of this subunit from the native complex. The implications of the nitrite reductase structure for understanding multi-electron processes are discussed in terms of differences in the protein environments of the cofactors.

  • Reactions of spinach nitrite reductase with its substrate, nitrite, and a putative intermediate, hydroxylamine.
    Biochemistry, 2004
    Co-Authors: Sofya Kuznetsova, Pierre Sétif, Masakazu Hirasawa, David B. Knaff, Tony A Mattioli
    Abstract:

    Plant nitrite reductase (NiR) catalyzes the reduction of nitrite (NO2-) to ammonia, using reduced ferredoxin as the electron donor. NiR contains a [4Fe-4S] cluster and an Fe-Siroheme, which is the nitrite binding site. In the enzyme's as-isolated form ([4Fe-4S]2+/Fe3+), resonance Raman spectroscopy indicated that the Siroheme is in the high-spin ferric hexacoordinated state with a weak sixth axial ligand. Kinetic and spectroscopic experiments showed that the reaction of NiR with NO2- results in an unexpectedly EPR-silent complex formed in a single step with a rate constant of 0.45 ± 0.01 s-1. This binding rate is slow compared to that expected from the NiR turnover rates reported in the literature, suggesting that binding of NO2- to the as-isolated form of NiR is not the predominant type of substrate binding during enzyme turnover. Resonance Raman spectroscopic characterization of this complex indicated that (i) the Siroheme iron is low-spin hexacoordinated ferric, (ii) the ligand coordination is unusuall...

  • Oxidation-reduction properties of maize ferredoxin: sulfite oxidoreductase.
    Biochimica et biophysica acta, 2004
    Co-Authors: Masakazu Hirasawa, Toshiharu Hase, Masato Nakayama, David B. Knaff
    Abstract:

    Oxidation-reduction titrations have been carried out on the wild-type, ferredoxin-dependent sulfite reductase from maize and two site-specific variants of the enzyme. E(m) values have been determined for the Siroheme and [4Fe-4S] cluster prosthetic groups of the enzyme, which titrate as independent, one-electron carriers. Visible-region difference spectra suggest that reduction of the [4Fe-4S] cluster significantly perturbs the spectrum of the reduced Siroheme group of the enzyme. The effects of Siroheme axial ligation, by either cyanide or phosphate ligands, on the redox properties of sulfite reductase have also been examined. For comparison, the effects of phosphate and cyanide on the redox properties of the ferredoxin-dependent nitrite reductase of spinach chloroplasts, an enzyme with the same prosthetic group arrangement as sulfite reductase, have been examined.