Light-driven carbon-carbon bond formation via CO2 reduction catalyzed by complexes of CdS nanorods and a 2-oxoacid oxidoreductase.

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.

An S=1 Iron(IV) Intermediate Revealed in a Non-Heme Iron Enzyme-Catalyzed Oxidative C-S Bond Formation.

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.

Elucidating Electron Storage and Distribution within the Pentaheme Scaffold of Cytochrome c Nitrite Reductase (NrfA).

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.

Mechanism of Reduction of an Aminyl Radical Intermediate in the Radical SAM GTP 3',8-Cyclase MoaA.

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'•) trapped in the active site revealed isotropic exchange interaction (∼260 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.

Metalloprotein switches that display chemical-dependent electron transfer in cells.

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.

A Stable Ferryl Porphyrin at the Active Site of Y463M BthA.

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)═O 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)═O 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.

Structural Properties and Catalytic Implications of the SPASM Domain Iron-Sulfur Clusters in Methylorubrum extorquens PqqE.

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.

Spectroscopic and Electrochemical Characterization of the Mycofactocin Biosynthetic Protein, MftC, Provides Insight into Its Redox Flipping Mechanism.

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.

Deconvolution of reduction potentials of formate dehydrogenase from Cupriavidus necator.

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.

Resonance Raman, Electron Paramagnetic Resonance, and Magnetic Circular Dichroism Spectroscopic Investigation of Diheme Cytochrome c Peroxidases from Nitrosomonas europaea and Shewanella oneidensis.

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 FeIV═O intermediate.

Deconvoluting the Reduction Potentials for the Three [4Fe-4S] Clusters in an AdoMet Radical SCIFF Maturase.

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.

Influence of heme c attachment on heme conformation and potential.

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.

Molecular basis of cobalamin-dependent RNA modification.

Queuosine (Q) was discovered in the wobble position of a transfer RNA (tRNA) 47 years ago, yet the final biosynthetic enzyme responsible for Q-maturation, epoxyqueuosine (oQ) reductase (QueG), was only recently identified. QueG is a cobalamin (Cbl)-dependent, [4Fe-4S] cluster-containing protein that produces the hypermodified nucleoside Q in situ on four tRNAs. To understand how QueG is able to perform epoxide reduction, an unprecedented reaction for a Cbl-dependent enzyme, we have determined a series of high resolution structures of QueG from Bacillus subtilis Our structure of QueG bound to a tRNATyr anticodon stem loop shows how this enzyme uses a HEAT-like domain to recognize the appropriate anticodons and position the hypermodified nucleoside into the enzyme active site. We find Q bound directly above the Cbl, consistent with a reaction mechanism that involves the formation of a covalent Cbl-tRNA intermediate. Using protein film electrochemistry, we show that two [4Fe-4S] clusters adjacent to the Cbl have redox potentials in the range expected for Cbl reduction, suggesting how Cbl can be activated for nucleophilic attack on oQ. Together, these structural and electrochemical data inform our understanding of Cbl dependent nucleic acid modification.

Structures of benzylsuccinate synthase elucidate roles of accessory subunits in glycyl radical enzyme activation and activity.

Anaerobic degradation of the environmental pollutant toluene is initiated by the glycyl radical enzyme benzylsuccinate synthase (BSS), which catalyzes the radical addition of toluene to fumarate, forming benzylsuccinate. We have determined crystal structures of the catalytic α-subunit of BSS with its accessory subunits ß and γ, which both bind a [4Fe-4S] cluster and are essential for BSS activity in vivo. We find that BSSα has the common glycyl radical enzyme fold, a 10-stranded ß/α-barrel that surrounds the glycyl radical cofactor and active site. Both accessory subunits ß and γ display folds related to high potential iron-sulfur proteins but differ substantially from each other in how they interact with the α-subunit. BSSγ binds distally to the active site, burying a hydrophobic region of BSSα, whereas BSSß binds to a hydrophilic surface of BSSα that is proximal to the active site. To further investigate the function of BSSß, we determined the structure of a BSSαγ complex. Remarkably, we find that the barrel partially opens, allowing the C-terminal region of BSSα that houses the glycyl radical to shift within the barrel toward an exit pathway. The structural changes that we observe in the BSSαγ complex center around the crucial glycyl radical domain, thus suggesting a role for BSSß in modulating the conformational dynamics required for enzyme activity. Accompanying proteolysis experiments support these structural observations.

Functionally Distinct Bacterial Cytochrome c Peroxidases Proceed through a Common (Electro)catalytic Intermediate.

The diheme cytochrome c peroxidase from Shewanella oneidensis (So CcP) requires a single electron reduction to convert the oxidized, as-isolated enzyme to an active conformation. We employ protein film voltammetry to investigate the mechanism of hydrogen peroxide turnover by So CcP. When the enzyme is poised in the active state by incubation with sodium l-ascorbate, the graphite electrode specifically captures a highly active state that turns over peroxide in a high potential regime. This is the first example of an on-pathway catalytic intermediate observed for a bacterial diheme cytochrome c peroxidase that requires reductive activation, consistent with the observed voltammetric response from the diheme cytochrome c peroxidase from Nitrosomonas europaea (Ne), which is constitutively active and does not require the same one electron activation. Mutational analysis at the active site of So CcP confirms that the rate-limiting step involves a proton-coupled single electron reduction of a high valent iron species centered on the low-potential heme, consistent with the same mutation in Ne CcP. The pH dependence of catalysis for wild-type So CcP suggests that reduction shifts the pK(a)'s of at least two amino acids. Mutation of His81 in "loop 1", a surface exposed loop thought to shift conformation during the reductive activation process, eliminated one of the pH dependent features, confirming that the loop 1 shifts, changing the environment of His81 during the rate-limiting step. The observed catalytic intermediate has the same electron stoichiometry and similar pH dependence to that previously reported for Ne CcP, which is constitutively active and therefore hypothesized to follow a different catalytic mechanism. The prominent similarities between the rate-limiting steps of differing mechanistic classes of bCcPs suggest unexpected similarities in the intermediates formed.

Transformations of the FeS Clusters of the Methylthiotransferases MiaB and RimO, Detected by Direct Electrochemistry.

The methylthiotransferases (MTTases) represent a subfamily of the S-adenosylmethionine (AdoMet) radical superfamily of enzymes that catalyze the attachment of a methylthioether (-SCH3) moiety on unactivated carbon centers. These enzymes contain two [4Fe-4S] clusters, one of which participates in the reductive fragmentation of AdoMet to generate a 5'-deoxyadenosyl 5'-radical and the other of which, termed the auxiliary cluster, is believed to play a central role in constructing the methylthio group and attaching it to the substrate. Because the redox properties of the bound cofactors within the AdoMet radical superfamily are so poorly understood, we have examined two MTTases in parallel, MiaB and RimO, using protein electrochemistry. We resolve the redox potentials of each [4Fe-4S] cluster, show that the auxiliary cluster has a potential higher than that of the AdoMet-binding cluster, and demonstrate that upon incubation of either enzyme with AdoMet, a unique low-potential state of the enzyme emerges. Our results are consistent with a mechanism whereby the auxiliary cluster is transiently methylated during substrate methylthiolation.

Spectroscopic and Electrochemical Characterization of the Iron-Sulfur and Cobalamin Cofactors of TsrM, an Unusual Radical S-Adenosylmethionine Methylase.

TsrM, an annotated radical S-adenosylmethionine (SAM) enzyme, catalyzes the methylation of carbon 2 of the indole ring of L-tryptophan. Its reaction is the first step in the biosynthesis of the unique quinaldic acid moiety of thiostrepton A, a thiopeptide antibiotic. The appended methyl group derives from SAM; however, the enzyme also requires cobalamin and iron-sulfur cluster cofactors for turnover. In this work we report the overproduction and purification of TsrM and the characterization of its metallocofactors by UV-visible, electron paramagnetic resonance, hyperfine sublevel correlation (HYSCORE), and Mössbauer spectroscopies as well as protein-film electrochemistry (PFE). The enzyme contains 1 equiv of its cobalamin cofactor in its as-isolated state and can be reconstituted with iron and sulfide to contain one [4Fe-4S] cluster with a site-differentiated Fe(2+)/Fe(3+) pair. Our spectroscopic studies suggest that TsrM binds cobalamin in an uncharacteristic five-coordinate base-off/His-off conformation, whereby the dimethylbenzimidazole group is replaced by a non-nitrogenous ligand, which is likely a water molecule. Electrochemical analysis of the protein by PFE indicates a one-electron redox feature with a midpoint potential of -550 mV, which is assigned to a [4Fe-4S](2+)/[4Fe-4S](+) redox couple. Analysis of TsrM by Mössbauer and HYSCORE spectroscopies suggests that SAM does not bind to the unique iron site of the cluster in the same manner as in other radical SAM (RS) enzymes, yet its binding still perturbs the electronic configuration of both the Fe/S cluster and the cob(II)alamin cofactors. These biophysical studies suggest that TsrM is an atypical RS enzyme, consistent with its reported inability to catalyze formation of a 5'-deoxyadenosyl 5'-radical.

A Ferredoxin Disulfide Reductase Delivers Electrons to the Methanosarcina barkeri Class III Ribonucleotide Reductase.

Two subtypes of class III anaerobic ribonucleotide reductases (RNRs) studied so far couple the reduction of ribonucleotides to the oxidation of formate, or the oxidation of NADPH via thioredoxin and thioredoxin reductase. Certain methanogenic archaea contain a phylogenetically distinct third subtype of class III RNR, with distinct active-site residues. Here we report the cloning and recombinant expression of the Methanosarcina barkeri class III RNR and show that the electrons required for ribonucleotide reduction can be delivered by a [4Fe-4S] protein ferredoxin disulfide reductase, and a conserved thioredoxin-like protein NrdH present in the RNR operon. The diversity of class III RNRs reflects the diversity of electron carriers used in anaerobic metabolism.

Correlations between the Electronic Properties of Shewanella oneidensis Cytochrome c Nitrite Reductase (ccNiR) and Its Structure: Effects of Heme Oxidation State and Active Site Ligation.

The electrochemical properties of Shewanella oneidensis cytochrome c nitrite reductase (ccNiR), a homodimer that contains five hemes per protomer, were investigated by UV-visible and electron paramagnetic resonance (EPR) spectropotentiometries. Global analysis of the UV-vis spectropotentiometric results yielded highly reproducible values for the heme midpoint potentials. These midpoint potential values were then assigned to specific hemes in each protomer (as defined in previous X-ray diffraction studies) by comparing the EPR and UV-vis spectropotentiometric results, taking advantage of the high sensitivity of EPR spectra to the structural microenvironment of paramagnetic centers. Addition of the strong-field ligand cyanide led to a 70 mV positive shift of the active site's midpoint potential, as the cyanide bound to the initially five-coordinate high-spin heme and triggered a high-spin to low-spin transition. With cyanide present, three of the remaining hemes gave rise to distinctive and readily assignable EPR spectral changes upon reduction, while a fourth was EPR-silent. At high applied potentials, interpretation of the EPR spectra in the absence of cyanide was complicated by a magnetic interaction that appears to involve three of five hemes in each protomer. At lower applied potentials, the spectra recorded in the presence and absence of cyanide were similar, which aided global assignment of the signals. The midpoint potential of the EPR-silent heme could be assigned by default, but the assignment was also confirmed by UV-vis spectropotentiometric analysis of the H268M mutant of ccNiR, in which one of the EPR-silent heme's histidine axial ligands was replaced with a methionine.

Electrochemical Resolution of the [4Fe-4S] Centers of the AdoMet Radical Enzyme BtrN: Evidence of Proton Coupling and an Unusual, Low-Potential Auxiliary Cluster.

The S-adenosylmethionine (AdoMet) radical superfamily of enzymes includes over 113,500 unique members, each of which contains one indispensable iron-sulfur (FeS) cluster that is required to generate a 5'-deoxyadenosyl 5'-radical intermediate during catalysis. Enzymes within several subgroups of the superfamily, however, have been found to contain one or more additional FeS clusters. While these additional clusters are absolutely essential for enzyme activity, their exact roles in the function and/or mechanism of action of many of the enzymes are at best speculative, indicating a need to develop methods to characterize and study these clusters in more detail. Here, BtrN, an AdoMet radical dehydrogenase that catalyzes the two-electron oxidation of 2-deoxy-scyllo-inosamine to amino-dideoxy-scyllo-inosose, an intermediate in the biosynthesis of 2-deoxystreptamine antibiotics, is examined through direct electrochemistry, where the potential of both its AdoMet radical and auxiliary [4Fe-4S] clusters can be measured simultaneously. We find that the AdoMet radical cluster exhibits a midpoint potential of -510 mV, while the auxiliary cluster exhibits a midpoint potential of -765 mV, to our knowledge the lowest [4Fe-4S](2+/+) potential to be determined to date. The impact of AdoMet binding and the pH dependence of catalysis are also quantitatively observed. These data show that direct electrochemical methods can be used to further elucidate the chemistry of the burgeoning AdoMet radical superfamily in the future.