RESUMEN
Methanogens are a diverse group of Archaea that obligately couple energy conservation to the production of methane. Some methanogens encode alternate pathways for energy conservation, like anaerobic respiration, but the biochemical details of this process are unknown. We show that a multiheme c-type cytochrome called MmcA from Methanosarcina acetivorans is important for intracellular electron transport during methanogenesis and can also reduce extracellular electron acceptors like soluble Fe3+ and anthraquinone-2,6-disulfonate. Consistent with these observations, MmcA displays reversible redox features ranging from -100 to -450 mV versus SHE. Additionally, mutants lacking mmcA have significantly slower Fe3+ reduction rates. The mmcA locus is prevalent in members of the Order Methanosarcinales and is a part of a distinct clade of multiheme cytochromes that are closely related to octaheme tetrathionate reductases. Taken together, MmcA might act as an electron conduit that can potentially support a variety of energy conservation strategies that extend beyond methanogenesis.
Asunto(s)
Electrones , Methanosarcina , Transporte de Electrón , Methanosarcina/metabolismo , Oxidación-Reducción , Citocromos/metabolismo , Metano/metabolismoRESUMEN
Ergothioneine (ESH) and ovothiol A (OSHA) are two natural thiol-histidine derivatives. ESH has been implicated as a longevity vitamin and OSHA inhibits the proliferation of hepatocarcinoma. The key biosynthetic step of ESH and OSHA in the aerobic pathways is the O2 -dependent C-S bond formation catalyzed by non-heme iron enzymes (e.g., OvoA in ovothiol biosynthesis), but due to the lack of identification of key reactive intermediate the mechanism of this novel reaction is unresolved. In this study, we report the identification and characterization of a kinetically competent S=1 iron(IV) intermediate supported by a four-histidine ligand environment (three from the protein residues and one from the substrate) in enabling C-S bond formation in OvoA from Methyloversatilis thermotoleran, which represents the first experimentally observed intermediate spin iron(IV) species in non-heme iron enzymes. Results reported in this study thus set the stage to further dissect the mechanism of enzymatic oxidative C-S bond formation in the OSHA biosynthesis pathway. They also afford new opportunities to study the structure-function relationship of high-valent iron intermediates supported by a histidine rich ligand environment.
Asunto(s)
Histidina , Hierro , Histidina/metabolismo , Ligandos , Catálisis , Estrés OxidativoRESUMEN
Methanogens are a diverse group of Archaea that couple energy conservation to the production of methane gas. While most methanogens have no alternate mode of energy conservation, strains like Methanosarcina acetivorans are known to also conserve energy by dissimilatory metal reduction (DSMR) in the presence of soluble ferric iron or iron-containing minerals. The ecological ramifications of energy conservation decoupled from methane production in methanogens are substantial, yet the molecular details are poorly understood. In this work, we conducted in vitro and in vivo studies with a multiheme c-type cytochrome (MHC), called MmcA, to establish its role during methanogenesis and DSMR in M. acetivorans. MmcA purified from M. acetivorans can donate electrons to methanophenazine, a membrane-bound electron carrier, to facilitate methanogenesis. In addition, MmcA can also reduce Fe(III) and the humic acid analog anthraquinone-2,6-disulfonate (AQDS) during DSMR. Furthermore, mutants lacking mmcA have slower Fe(III) reduction rates. The redox reactivities of MmcA are consistent with the electrochemical data where MmcA displays reversible redox features ranging from -100 to -450 mV versus SHE. MmcA is prevalent in members of the Order Methanosarcinales but does not belong to a known family of MHCs linked to extracellular electron transfer, bioinformatically, and instead forms a distinct clade that is closely related to octaheme tetrathionate reductases. Taken together, this study shows that MmcA is widespread in methanogens with cytochromes where it acts as an electron conduit to support a variety of energy conservation strategies that extend beyond methanogenesis.
RESUMEN
Rieske-type non-heme iron oxygenases/oxidases catalyze a wide range of transformations. Their applications in bioremediation or biocatalysis face two key barriers: the need of expensive NAD(P)H as a reductant and a proper reductase to mediate the electron transfer from NAD(P)H to the oxygenases. To bypass the need of both the reductase and NAD(P)H, using Rieske-type oxygenase (Stc2) catalyzed oxidative demethylation as the model system, we report Stc2 photocatalysis using eosin Y/sulfite as the photosensitizer/sacrificial reagent pair. In a flow-chemistry setting to separate the photo-reduction half-reaction and oxidation half-reaction, Stc2 photo-biocatalysis outperforms the Stc2-NAD(P)H-reductase (GbcB) system. In addition, in a few other selected Rieske enzymes (NdmA, CntA, and GbcA), and a flavin-dependent enzyme (iodotyrosine deiodinase, IYD), the eosin Y/sodium sulfite photo-reduction pair could also serve as the NAD(P)H-reductase surrogate to support catalysis, which implies the potential applicability of this photo-reduction system to other redox enzymes.
RESUMEN
The diversity of the reactions catalyzed by radical S-adenosyl-l-methionine (SAM) enzymes is achieved at least in part through the variety of mechanisms to quench their radical intermediates. In the SPASM-twitch family, the largest family of radical SAM enzymes, the radical quenching step is thought to involve an electron transfer to or from an auxiliary 4Fe-4S cluster in or adjacent to the active site. However, experimental demonstration of such functions remains limited. As a representative member of this family, MoaA has one radical SAM cluster ([4Fe-4S]RS) and one auxiliary cluster ([4Fe-4S]AUX), and catalyzes a unique 3',8-cyclization of GTP into 3',8-cyclo-7,8-dihydro-GTP (3',8-cH2GTP) in the molybdenum cofactor (Moco) biosynthesis. Here, we report a mechanistic investigation of the radical quenching step in MoaA, a chemically challenging reduction of 3',8-cyclo-GTP-N7 aminyl radical. We first determined the reduction potentials of [4Fe-4S]RS and [4Fe-4S]AUX as -510 mV and -455 mV, respectively, using a combination of protein film voltammogram (PFV) and electron paramagnetic resonance (EPR) spectroscopy. Subsequent Q-band EPR characterization of 5'-deoxyadenosine C4' radical (5'-dA-C4'â¢) trapped in the active site revealed isotropic exchange interaction (â¼260 MHz) between 5'-dA-C4'⢠and [4Fe-4S]AUX1+, suggesting that [4Fe-4S]AUX is in the reduced (1+) state during the catalysis. Together with density functional theory (DFT) calculation, we propose that the aminyl radical reduction proceeds through a proton-coupled electron transfer (PCET), where [4Fe-4S]AUX serves as an electron donor and R17 residue acts as a proton donor. These results provide detailed mechanistic insights into the radical quenching step of radical SAM enzyme catalysis.
Asunto(s)
Radicales Libres/química , Guanosina Trifosfato/metabolismo , Hidrolasas/metabolismo , S-Adenosilmetionina/metabolismo , Dominio Catalítico , Teoría Funcional de la Densidad , Espectroscopía de Resonancia por Spin del Electrón , Transporte de Electrón , Guanosina Trifosfato/química , Hidrolasas/genética , Simulación de Dinámica Molecular , Mutagénesis , S-Adenosilmetionina/química , Staphylococcus aureus/enzimologíaRESUMEN
Cytochrome c nitrite reductases (CcNIR or NrfA) play important roles in the global nitrogen cycle by conserving the usable nitrogen in the soil. Here, the electron storage and distribution properties within the pentaheme scaffold of Geobacter lovleyi NrfA were investigated via electron paramagnetic resonance (EPR) spectroscopy coupled with chemical titration experiments. Initially, a chemical reduction method was established to sequentially add electrons to the fully oxidized protein, 1 equiv at a time. The step-by-step reduction of the hemes was then followed using ultraviolet-visible absorption and EPR spectroscopy. EPR spectral simulations were used to elucidate the sequence of heme reduction within the pentaheme scaffold of NrfA and identify the signals of all five hemes in the EPR spectra. Electrochemical experiments ascertain the reduction potentials for each heme, observed in a narrow range from +10 mV (heme 5) to -226 mV (heme 3) (vs the standard hydrogen electrode). On the basis of quantitative analysis and simulation of the EPR data, we demonstrate that hemes 4 and 5 are reduced first (before the active site heme 1) and serve the purpose of an electron storage unit within the protein. To probe the role of the central heme 3, an H108M NrfA variant was generated where the reduction potential of heme 3 is shifted positively (from -226 to +48 mV). The H108M mutation significantly impacts the distribution of electrons within the pentaheme scaffold and the reduction potentials of the hemes, reducing the catalytic activity of the enzyme to 1% compared to that of the wild type. We propose that this is due to heme 3's important role as an electron gateway in the wild-type enzyme.
Asunto(s)
Grupo Citocromo c/metabolismo , Citocromos a1/metabolismo , Citocromos c1/metabolismo , Geobacter/metabolismo , Nitrato Reductasas/metabolismo , Dominio Catalítico , Cristalografía por Rayos X/métodos , Grupo Citocromo c/química , Citocromos a1/química , Citocromos c1/química , Espectroscopía de Resonancia por Spin del Electrón/métodos , Electrones , Geobacter/química , Hemo/química , Hemo/metabolismo , Modelos Moleculares , Nitrato Reductasas/química , Nitrito Reductasas/química , Nitrito Reductasas/metabolismo , Oxidación-Reducción , Conformación ProteicaRESUMEN
Understanding the relationship between the metallocofactor and its protein environment is the key to uncovering the mechanism of metalloenzymes. PqqE, a radical S-adenosylmethionine enzyme in pyrroloquinoline quinone (PQQ) biosynthesis, contains three iron-sulfur cluster binding sites. Two auxiliary iron-sulfur cluster binding sites, designated as AuxI and AuxII, use distinctive ligands compared to other proteins in the family while their functions remain unclear. Here, we investigate the electronic properties of these iron-sulfur clusters and compare the catalytic efficiency of wild-type (WT) Methylorubrum extorquens AM1 PqqE to a range of mutated constructs. Using native mass spectrometry, protein film electrochemistry, and electron paramagnetic resonance spectroscopy, we confirm the previously proposed incorporation of a mixture of [2Fe-2S] and [4Fe-4S] clusters at the AuxI site and are able to assign redox potentials to each of the three iron-sulfur clusters. Significantly, a conservative mutation at AuxI, C268H, shown to selectively incorporate a [4Fe-4S] cluster, catalyzes an enhancement of uncoupled S-adenosylmethionine cleavage relative to WT, together with the elimination of detectable peptide cross-linked product. While a [4Fe-4S] cluster can be tolerated at the AuxI site, the aggregate findings suggest a functional [2Fe-2S] configuration within the AuxI site. PqqE variants with nondestructive ligand replacements at AuxII also show that the reduction potential at this site can be manipulated by changing the electronegativity of the unique aspartate ligand. A number of novel mechanistic features are proposed based on the kinetic and spectroscopic data. Additionally, bioinformatic analyses suggest that the unique ligand environment of PqqE may be relevant to its role in PQQ biosynthesis within an oxygen-dependent biosynthetic pathway.
Asunto(s)
Proteínas Bacterianas/metabolismo , Endopeptidasas/metabolismo , Hierro/metabolismo , Methylobacterium extorquens/química , Azufre/metabolismo , Proteínas Bacterianas/química , Biocatálisis , Cristalografía por Rayos X , Endopeptidasas/química , Hierro/química , Methylobacterium extorquens/metabolismo , Modelos Moleculares , Estructura Molecular , Azufre/químicaRESUMEN
BthA is a diheme enzyme that is a member of the bacterial cytochrome c peroxidase superfamily, capable of generating a highly unusual Fe(IV)Fe(IV)âO oxidation state, known to be responsible for long-range oxidative chemistry in the enzyme MauG. Here, we show that installing a canonical Met ligand in lieu of the Tyr found at the heme of MauG associated with electron transfer, results in a construct that yields an unusually stable Fe(IV)âO porphyrin at the peroxidatic heme. This state is spontaneously formed at ambient conditions using either molecular O2 or H2O2. The resulting data illustrate how a ferryl iron, with unforeseen stability, may be achieved in biology.
Asunto(s)
Proteínas Bacterianas/metabolismo , Citocromo-c Peroxidasa/metabolismo , Hierro/metabolismo , Porfirinas/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Sitios de Unión , Citocromo-c Peroxidasa/química , Citocromo-c Peroxidasa/genética , Hierro/química , Modelos Moleculares , Mutación , Porfirinas/químicaRESUMEN
Redox enzymes are capable of catalyzing a vast array of useful reactions, but they require redox partners that donate or accept electrons. Semiconductor nanocrystals provide a mechanism to convert absorbed photon energy into redox equivalents for enzyme catalysis. Here, we describe a system for photochemical carbon-carbon bond formation to make 2-oxoglutarate by coupling CO2 with a succinyl group. Photoexcited electrons from cadmium sulfide nanorods (CdS NRs) transfer to 2-oxoglutarate:ferredoxin oxidoreductase from Magnetococcus marinus MC-1 (MmOGOR), which catalyzes a carbon-carbon bond formation reaction. We thereby decouple MmOGOR from its native role in the reductive tricarboxylic acid cycle and drive it directly with light. We examine the dependence of 2-oxoglutarate formation on a variety of factors and, using ultrafast transient absorption spectroscopy, elucidate the critical role of electron transfer (ET) from CdS NRs to MmOGOR. We find that the efficiency of this ET depends strongly on whether the succinyl CoA (SCoA) cosubstrate is bound at the MmOGOR active site. We hypothesize that the conformational changes due to SCoA binding impact the CdS NR-MmOGOR interaction in a manner that decreases ET efficiency compared to the enzyme with no cosubstrate bound. Our work reveals structural considerations for the nano-bio interfaces involved in light-driven enzyme catalysis and points to the competing factors of enzyme catalysis and ET efficiency that may arise when complex enzyme reactions are driven by artificial light absorbers.
Asunto(s)
Compuestos de Cadmio/química , Dióxido de Carbono/metabolismo , Carbono/química , Luz , Nanotubos/química , Oxidorreductasas/metabolismo , Fotoquímica/métodos , Sulfuros/química , Acilcoenzima A , Alphaproteobacteria/enzimología , Catálisis , Ciclo del Ácido Cítrico , Transporte de Electrón , Electrones , Ferredoxinas/metabolismo , Cetoácidos , Ácidos Cetoglutáricos/metabolismo , Nanopartículas/química , Oxidación-ReducciónRESUMEN
The formate dehydrogenase enzyme from Cupriavidus necator (FdsABG) carries out the two-electron oxidation of formate to CO2, but is also capable of reducing CO2 back to formate, a potential biofuel. FdsABG is a heterotrimeric enzyme that performs this transformation using nine redox-active cofactors: a bis(molybdopterin guanine dinucleotide) (bis-MGD) at the active site coupled to seven iron-sulfur clusters, and one equivalent of flavin mononucleotide (FMN). To better understand the pathway of electron flow in FdsABG, the reduction potentials of the various cofactors were examined through direct electrochemistry. Given the redundancy of cofactors, a truncated form of the FdsA subunit was developed that possesses only the bis-MGD active site and a singular [4Fe-4S] cluster. Electrochemical characterization of FdsABG compared to truncated FdsA shows that the measured reduction potentials are remarkably similar despite the truncation with two observable features at - 265 mV and - 455 mV vs SHE, indicating that the voltammetry of the truncated enzyme is representative of the reduction potentials of the intact heterotrimer. By producing truncated FdsA without the necessary maturation factors required for bis-MGD insertion, a form of the truncated FdsA that possesses only the [4Fe-4S] was produced, which gives a single voltammetric feature at - 525 mV, allowing the contributions of the molybdenum cofactor to be associated with the observed feature at - 265 mV. This method allowed for the deconvolution of reduction potentials for an enzyme with highly complex cofactor content to know more about the thermodynamic landscape of catalysis.
Asunto(s)
Cupriavidus necator/enzimología , Cupriavidus necator/metabolismo , Formiato Deshidrogenasas/metabolismo , Catálisis , Coenzimas/metabolismo , Cupriavidus necator/genética , Mononucleótido de Flavina/metabolismo , Formiato Deshidrogenasas/química , Formiato Deshidrogenasas/genética , Proteínas Hierro-Azufre/química , Proteínas Hierro-Azufre/genética , Proteínas Hierro-Azufre/metabolismo , Metaloproteínas/metabolismo , Cofactores de Molibdeno , Oxidación-Reducción , Pteridinas/metabolismoRESUMEN
2-oxoglutarate:ferredoxin oxidoreductase (OGOR) is a thiamine pyrophosphate (TPP) and [4Fe-4S] cluster-dependent enzyme from the reductive tricarboxylic acid (rTCA) cycle that fixes CO2 to succinyl-CoA, forming 2-oxoglutarate and CoA. Here we report an OGOR from the rTCA cycle of Magnetococcus marinus MC-1, along with all three potential ferredoxin (Fd) redox partners. We demonstrate MmOGOR operates bidirectionally (both CO2-fixing and 2-oxoglutarate oxidizing), and that only one Fd (MmFd1) supports efficient catalysis. Our 1.94-Å and 2.80-Å resolution crystal structures of native and substrate-bound forms of MmOGOR reveal the determinants of substrate specificity and CoA-binding in an OGOR, and illuminate the [4Fe-4S] cluster environment, portraying the electronic conduit allowing MmFd1 to be wired to the bound-TPP. Structural and biochemical data further identify Glu45α as a mobile residue that impacts catalytic bias toward CO2-fixation although it makes no direct contact with TPP-bound intermediates, indicating that reaction directionality can be tuned by second layer interactions. (149 of 150 words limit).
RESUMEN
Bacterial diheme peroxidases represent a diverse enzyme family with functions that range from hydrogen peroxide (H2O2) reduction to post-translational modifications. By implementing a sequence similarity network (SSN) of the bCCP_MauG superfamily, we present the discovery of a unique diheme peroxidase BthA conserved in all Burkholderia. Using a combination of magnetic resonance, near-IR and Mössbauer spectroscopies and electrochemical methods, we report that BthA is capable of generating a bis-Fe(IV) species previously thought to be a unique feature of the diheme enzyme MauG. However, BthA is not MauG-like in that it catalytically converts H2O2 to water, and a 1.54-Å resolution crystal structure reveals striking differences between BthA and other superfamily members, including the essential residues for both bis-Fe(IV) formation and H2O2 turnover. Taken together, we find that BthA represents a previously undiscovered class of diheme enzymes, one that stabilizes a bis-Fe(IV) state and catalyzes H2O2 turnover in a mechanistically distinct manner.
Asunto(s)
Proteínas Bacterianas/metabolismo , Burkholderia/enzimología , Hemoproteínas/metabolismo , Peroxidasas/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Burkholderia/genética , Dominio Catalítico , Cristalografía por Rayos X , Estabilidad de Enzimas , Hemoproteínas/química , Hemoproteínas/genética , Hierro/química , Cinética , Modelos Moleculares , Oxidación-Reducción , Peroxidasas/química , Peroxidasas/genética , Conformación ProteicaRESUMEN
Mycofactocin is a putative redox cofactor and is classified as a ribosomally synthesized and post-translationally modified peptide (RiPP). Some RiPP natural products, including mycofactocin, rely on a radical S-adenosylmethionine (RS, SAM) protein to modify the precursor peptide. Mycofactocin maturase, MftC, is a unique RS protein that catalyzes the oxidative decarboxylation and C-C bond formation on the precursor peptide MftA. However, the number, chemical nature, and catalytic roles for the MftC [Fe-S] clusters remain unknown. Here, we report that MftC binds a RS [4Fe-4S] cluster and two auxiliary [4Fe-4S] clusters that are required for MftA modification. Furthermore, electron paramagnetic resonance spectra of MftC suggest that SAM and MftA affect the environments of the RS and Aux I cluster, whereas the Aux II cluster is unaffected by the substrates. Lastly, reduction potential assignments of individual [4Fe-4S] clusters by protein film voltammetry show that their potentials are within 100 mV of each other.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Proteínas Hierro-Azufre/química , Proteínas Hierro-Azufre/metabolismo , Proteínas Bacterianas/genética , Catálisis , Dominio Catalítico , Cisteína/química , Técnicas Electroquímicas , Espectroscopía de Resonancia por Spin del Electrón , Proteínas Hierro-Azufre/genética , Mycobacterium ulcerans/química , Oxidación-Reducción , S-Adenosilmetionina/metabolismo , Espectroscopía de MossbauerRESUMEN
Biological electron transfer is challenging to directly regulate using environmental conditions. To enable dynamic, protein-level control over energy flow in metabolic systems for synthetic biology and bioelectronics, we created ferredoxin logic gates that utilize transcriptional and post-translational inputs to control energy flow through a synthetic electron transfer pathway that is required for bacterial growth. These logic gates were created by subjecting a thermostable, plant-type ferredoxin to backbone fission and fusing the resulting fragments to a pair of proteins that self-associate, a pair of proteins whose association is stabilized by a small molecule, and to the termini of a ligand-binding domain. We show that the latter domain insertion design strategy yields an allosteric ferredoxin switch that acquires an oxygen-tolerant [2Fe-2S] cluster and can use different chemicals, including a therapeutic drug and an environmental pollutant, to control the production of a reduced metabolite in Escherichia coli and cell lysates.
Asunto(s)
Transporte de Electrón/fisiología , Metaloproteínas/fisiología , Secuencia de Aminoácidos , Espectroscopía de Resonancia por Spin del Electrón/métodos , Transporte de Electrón/efectos de los fármacos , Electrones , Escherichia coli/metabolismo , Ferredoxinas/fisiología , Metaloproteínas/genética , Mutagénesis Sitio-Dirigida/métodos , Procesamiento Proteico-Postraduccional/fisiologíaRESUMEN
Most organisms contain multiple soluble protein-based redox carriers such as members of the ferredoxin (Fd) family, that contain one or more iron-sulfur clusters. The potential redundancy of Fd proteins is poorly understood, particularly in connection to the ability of Fd proteins to deliver reducing equivalents to members of the "radical SAM," or S-adenosylmethionine radical enzyme (ARE) superfamily, where the activity of all known AREs requires that an essential iron-sulfur cluster bound by the enzyme be reduced to the catalytically relevant [Fe4 S4 ]1+ oxidation state. As it is still unclear whether a single Fd in a given organism is specific to individual redox partners, we have examined the five Fd proteins found within Thermotoga maritima via direct electrochemistry, to compare them in a side-by-side fashion for the first time. While a single [Fe4 S4 ]-cluster bearing Fd (TM0927) has a potential of -420 mV, the other four 2x[Fe4 S4 ]-bearing Fds (TM1175, TM1289, TM1533, and TM1815) have potentials that vary significantly, including cases where the two clusters of the same Fd are essentially coincident (e.g., TM1175) and those where the potentials are well separate (TM1815).
Asunto(s)
Ferredoxinas/química , Thermotoga maritima/química , Ferredoxinas/metabolismo , Oxidación-Reducción , Thermotoga maritima/metabolismoRESUMEN
MiaB is a member of the methylthiotransferase subclass of the radical S-adenosylmethionine (SAM) superfamily of enzymes, catalyzing the methylthiolation of C2 of adenosines bearing an N6 -isopentenyl (i6 A) group found at position 37 in several tRNAs to afford 2-methylthio-N6 -(isopentenyl)adenosine (ms2 i6 A). MiaB uses a reduced [4Fe-4S]+ cluster to catalyze a reductive cleavage of SAM to generate a 5'-deoxyadenosyl 5'-radical (5'-dAâ¢)-a required intermediate in its reaction-as well as an additional [4Fe-4S]2+ auxiliary cluster. In Escherichia coli and many other organisms, re-reduction of the [4Fe-4S]2+ cluster to the [4Fe-4S]+ state is accomplished by the flavodoxin reducing system. Most mechanistic studies of MiaBs have been carried out on the enzyme from Thermotoga maritima (Tm), which lacks the flavodoxin reducing system, and which is not activated by E. coli flavodoxin. However, the genome of this organism encodes five ferredoxins (TM0927, TM1175, TM1289, TM1533, and TM1815), each of which might donate the requisite electron to MiaB and perhaps to other radical SAM enzymes. The genes encoding each of these ferredoxins were cloned, and the associated proteins were isolated and shown to support turnover by Tm MiaB. In addition, TM1639, the ferredoxin-NADP+ oxidoreductase subunit α (NfnA) from Tm was overproduced and isolated and shown to provide electrons to the Tm ferredoxins during Tm MiaB turnover. The resulting reactions demonstrate improved coupling between formation of the 5'-dA⢠and ms2 i6 A production, indicating that only one hydrogen atom abstraction is required for the reaction.
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Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimología , Sulfurtransferasas/metabolismo , Thermotoga maritima/enzimología , Transporte de Electrón/fisiología , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Ferredoxinas/genética , Ferredoxinas/metabolismo , Sulfurtransferasas/genética , Thermotoga maritima/genéticaRESUMEN
Cytochrome c peroxidases (bCcPs) are diheme enzymes required for the reduction of H2O2 to water in bacteria. There are two classes of bCcPs: one is active in the diferric form (constitutively active), and the other requires the reduction of the high-potential heme (H-heme) before catalysis commences (reductively activated) at the low-potential heme (L-heme). To improve our understanding of the mechanisms and heme electronic structures of these different bCcPs, a constitutively active bCcP from Nitrosomonas europaea ( NeCcP) and a reductively activated bCcP from Shewanella oneidensis ( SoCcP) were characterized in both the diferric and semireduced states by electron paramagnetic resonance (EPR), resonance Raman (rRaman), and magnetic circular dichroism (MCD) spectroscopy. In contrast to some previous crystallographic studies, EPR and rRaman spectra do not indicate the presence of significant amounts of a five-coordinate, high-spin ferric heme in NeCcP or SoCcP in either the diferric or semireduced state in solution. This observation points toward a mechanism of activation in which the active site L-heme is not in a static, five-coordinate state but where the activation is more subtle and likely involves formation of a six-coordinate hydroxo complex, which could then react with hydrogen peroxide in an acid-base-type reaction to create Compound 0, the ferric hydroperoxo complex. This mechanism lies in stark contrast to the diheme enzyme MauG that exhibits a static, five-coordinate open heme site at the peroxidatic heme and that forms a more stable FeIVâO intermediate.
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Dicroismo Circular/métodos , Citocromo-c Peroxidasa/química , Espectroscopía de Resonancia por Spin del Electrón/métodos , Hemo/química , Nitrosomonas europaea/enzimología , Oxidorreductasas/química , Shewanella/enzimología , Espectrometría Raman/métodos , Catálisis , Dominio Catalítico , Citocromo-c Peroxidasa/metabolismo , Hemo/metabolismo , Modelos Moleculares , Oxidación-Reducción , Oxidorreductasas/metabolismo , Conformación ProteicaRESUMEN
Enzymes in the S-adenosyl-l-methionine (AdoMet) radical enzyme superfamily are metalloenzymes that catalyze a wide variety of complex radical-mediated transformations with the aid of a [4Fe-4S] cluster, which is required for activation of AdoMet to generate the 5'-deoxyadenosyl radical to initiate the catalytic cycle. In addition to this cluster, some enzymes share an additional domain, the SPASM domain, that houses auxiliary FeS clusters whose functional significance is not clearly understood. The AdoMet radical enzyme Tte1186, which catalyzes a thioether cross-link in a cysteine rich peptide (SCIFF), has two auxiliary [4Fe-4S] clusters within a SPASM domain that are required for enzymatic activity but not for the generation of the 5'-deoxyadenosyl radical intermediate. Here we demonstrate the ability to measure independently the midpoint potentials of each of the three [4Fe-4S] clusters by employing Tte1186 variants for which only the first, second, or AdoMet binding cluster is bound. This allows, for the first time, assignment of reduction potentials for all clusters in an AdoMet radical enzyme with a SPASM domain. Our results show that the clusters have midpoint potentials that are within 100 mV of each other, suggesting that their electrochemical properties are not greatly influenced by the presence of the nearby clusters.
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Proteínas Bacterianas/química , Firmicutes/enzimología , Proteínas Hierro-Azufre/química , S-Adenosilmetionina/química , Secuencias de Aminoácidos , Dominios ProteicosRESUMEN
Heme c is characterized by its covalent attachment to a polypeptide. The attachment is typically to a CXXCH motif in which the two Cys form thioether bonds with the heme, "X" can be any amino acid other than Cys, and the His serves as a heme axial ligand. Some cytochromes c, however, contain heme attachment motifs with three or four intervening residues in a CX3CH or CX4CH motif. Here, the impacts of these variations in the heme attachment motif on heme ruffling and electronic structure are investigated by spectroscopically characterizing CX3CH and CX4CH variants of Hydrogenobacter thermophilus cytochrome c552. In addition, a novel CXCH variant is studied. 1H and 13C NMR, EPR, and resonance Raman spectra of the protein variants are analyzed to deduce the extent of ruffling using previously reported relationships between these spectral data and heme ruffling. In addition, the reduction potentials of these protein variants are measured using protein film voltammetry. The CXCH and CX4CH variants are found to have enhanced heme ruffling and lower reduction potentials. Implications of these results for the use of these noncanonical motifs in nature, and for the engineering of novel heme peptide structures, are discussed.
Asunto(s)
Grupo Citocromo c/química , Hemo/química , Bacterias/enzimología , Grupo Citocromo c/metabolismo , Hemo/análogos & derivados , Hemo/genética , Mutación , Conformación ProteicaRESUMEN
While protein film electrochemistry (PFE) has proven to be an effective tool in the interrogation of redox cofactors and assessing the electrocatalytic activity of many different enzymes, recently it has been proven to be useful for the study of the redox potentials of the cofactors of AdoMet radical enzymes (AREs). In this chapter, we review the challenges and opportunities of examining the redox cofactors of AREs in a high level of detail, particularly for the deconvolution of redox potentials of multiple cofactors. We comment on how to best assess the electroactive nature of any given ARE, and we see that when applied well, PFE allows for not only determining redox potentials, but also determining proton-coupling and ligand-binding phenomena in the ARE superfamily.