The Production of Nitrous Oxide by the Heme/Nonheme Diiron Center of Engineered Myoglobins (Fe_BMbs) Proceeds through a <i>trans</i>-Iron-Nitrosyl Dimer

Denitrifying NO reductases are transmembrane protein complexes that are evolutionarily related to heme/copper terminal oxidases. They utilize a heme/nonheme diiron center to reduce two NO molecules to N₂O. Engineering a nonheme Fe_B site within the heme distal pocket of sperm whale myoglobin has offered well-defined diiron clusters for the investigation of the mechanism of NO reduction in these unique active sites. In this study, we use FTIR spectroscopy to monitor the production of N₂O in solution and to show that the presence of a distal Fe_B^II is not sufficient to produce the expected product. However, the addition of a glutamate side chain peripheral to the diiron site allows for 50% of a productive single-turnover reaction. Unproductive reactions are characterized by resonance Raman spectroscopy as dinitrosyl complexes, where one NO molecule is bound to the heme iron to form a five-coordinate low-spin {FeNO}⁷ species with ν­(FeNO)_heme and ν­(NO)_heme at 522 and 1660 cm^–1, and a second NO molecule is bound to the nonheme Fe_B site with a ν­(NO)_FeB at 1755 cm^–1. Stopped-flow UV–vis absorption coupled with rapid-freeze-quench resonance Raman spectroscopy provide a detailed map of the reaction coordinates leading to the unproductive iron-nitrosyl dimer. Unexpectedly, NO binding to Fe_B is kinetically favored and occurs prior to the binding of a second NO to the heme iron, leading to a (six-coordinate low-spin heme-nitrosyl/Fe_B-nitrosyl) transient dinitrosyl complex with characteristic ν­(FeNO)_heme at 570 ± 2 cm^–1 and ν­(NO)_FeB at 1755 cm^–1. Without the addition of a peripheral glutamate, the dinitrosyl complex is converted to a dead-end product after the dissociation of the proximal histidine of the heme iron, but the added peripheral glutamate side chain in Fe_BMb2 lowers the rate of dissociation of the promixal histidine which in turn allows the (six-coordinate low-spin heme-nitrosyl/Fe_B-nitrosyl) transient dinitrosyl complex to decay with production of N₂O at a rate of 0.7 s^–1 at 4 °C. Taken together, our results support the proposed trans mechanism of NO reduction in NORs

Vibrational Analysis of Mononitrosyl Complexes in Hemerythrin and Flavodiiron Proteins: Relevance to Detoxifying NO Reductase

Flavodiiron proteins (FDPs) play important roles in the microbial nitrosative stress response in low-oxygen environments by reductively scavenging nitric oxide (NO). Recently, we showed that FMN-free diferrous FDP from <i>Thermotoga maritima</i> exposed to 1 equiv NO forms a stable diiron-mononitrosyl complex (deflavo-FDP­(NO)) that can react further with NO to form N₂O [Hayashi, T.; Caranto, J. D.; Wampler, D. A; Kurtz, D. M., Jr.; Moënne-Loccoz, P. Biochemistry 2010, 49, 7040−7049]. Here we report resonance Raman and low-temperature photolysis FTIR data that better define the structure of this diiron-mononitrosyl complex. We first validate this approach using the stable diiron-mononitrosyl complex of hemerythrin, Hr­(NO), for which we observe a ν­(NO) at 1658 cm^–1, the lowest ν­(NO) ever reported for a nonheme {FeNO}⁷ species. Both deflavo-FDP­(NO) and the mononitrosyl adduct of the flavinated FPD (FDP­(NO)) show ν­(NO) at 1681 cm^–1, which is also unusually low. These results indicate that, in Hr­(NO) and FDP­(NO), the coordinated NO is exceptionally electron rich, more closely approaching the Fe­(III)­(NO^–) resonance structure. In the case of Hr­(NO), this polarization may be promoted by steric enforcement of an unusually small FeNO angle, while in FDP­(NO), the Fe­(III)­(NO^–) structure may be due to a semibridging electrostatic interaction with the second Fe­(II) ion. In Hr­(NO), accessibility and steric constraints prevent further reaction of the diiron-mononitrosyl complex with NO, whereas in FDP­(NO) the increased nucleophilicity of the nitrosyl group may promote attack by a second NO to produce N₂O. This latter scenario is supported by theoretical modeling [Blomberg, L. M.; Blomberg, M. R.; Siegbahn, P. E. J. Biol. Inorg. Chem. 2007, 12, 79−89]. Published vibrational data on bioengineered models of denitrifying heme-nonheme NO reductases [Hayashi, T.; Miner, K. D.; Yeung, N.; Lin, Y.-W.; Lu, Y.; Moënne-Loccoz, P. Biochemistry 2011, 50, 5939−5947] support a similar mode of activation of a heme {FeNO}⁷ species by the nearby nonheme Fe­(II)

Direct Electrochemistry of Phanerochaete chrysosporium Cellobiose Dehydrogenase Covalently Attached onto Gold Nanoparticle Modified Solid Gold Electrodes

Achieving efficient electrochemical communication between redox enzymes and various electrode materials is one of the main challenges in bioelectrochemistry and is of great importance for developing electronic applications. Cellobiose dehydrogenase (CDH) is an extracellular flavocytochrome composed of a catalytic FAD containing dehydrogenase domain (DH_CDH), a heme <i>b</i> containing cytochrome domain (CYT_CDH), and a flexible linker region connecting the two domains. Efficient direct electron transfer (DET) of CDH from the basidiomycete Phanerochaete chrysosporium (<i>Pc</i>CDH) covalently attached to mixed self-assembled monolayer (SAM) modified gold nanoparticle (AuNP) electrode is presented. The thiols used were as follows: 4-aminothiophenol (4-ATP), 4-mercaptobenzoic acid (4-MBA), 4-mercaptophenol (4-MP), 11-mercapto-1-undecanamine (MUNH₂), 11-mercapto-1-undecanoic acid (MUCOOH), and 11-mercapto-1-undecanol (MUOH). A covalent linkage between <i>Pc</i>CDH and 4-ATP or MUNH₂ in the mixed SAMs was formed using glutaraldehyde as cross-linker. The covalent immobilization and the surface coverage of <i>Pc</i>CDH were confirmed with surface plasmon resonance (SPR). To improve current density, AuNPs were cast on the top of polycrystalline gold electrodes. For all the immobilized <i>Pc</i>CDH modified AuNPs electrodes, cyclic voltammetry exhibited clear electrochemical responses of the CYT_CDH with fast electron transfer (ET) rates in the absence of substrate (lactose), and the formal potential was evaluated to be +162 mV vs NHE at pH 4.50. The standard ET rate constant (<i>k</i>_s) was estimated for the first time for CDH and was found to be 52.1, 59.8, 112, and 154 s^–1 for 4-ATP/4-MBA, 4-ATP/4-MP, MUNH₂/MUCOOH, and MUNH₂/MUOH modified electrodes, respectively. At all the mixed SAM modified AuNP electrodes, <i>Pc</i>CDH showed DET only via the CYT_CDH. No DET communication between the DH_CDH domain and the electrode was found. The current density for lactose oxidation was remarkably increased by introduction of the AuNPs. The 4-ATP/4-MBA modified AuNPs exhibited a current density up to 30 μA cm^–2, which is ∼70 times higher than that obtained for a 4-ATP/4-MBA modified polycrystalline gold electrode. The results provide insight into fundamental electrochemical properties of CDH covalently immobilized on gold electrodes and promote further applications of CDHs for biosensors, biofuel cells, and bioelectrocatalysis

The Hemophore HasA from <i>Yersinia pestis</i> (HasA_yp) Coordinates Hemin with a Single Residue, Tyr75, and with Minimal Conformational Change

Hemophores from <i>Serratia marcescens</i> (HasA_sm) and <i>Pseudomonas aeruginosa</i> (HasA_p) bind hemin between two loops, which harbor the axial ligands H32 and Y75. Hemin binding to the Y75 loop triggers closing of the H32 loop and enables binding of H32. Because <i>Yersinia pestis</i> HasA (HasA_yp) presents a Gln at position 32, we determined the structures of apo- and holo-HasA_yp. Surprisingly, the Q32 loop in apo-HasA_yp is already in the closed conformation, but no residue from the Q32 loop binds hemin in holo-HasA_yp. In agreement with the minimal reorganization between the apo- and holo-structures, the hemin on-rate is too fast to detect by conventional stopped-flow measurements

A Nonheme, High-Spin {FeNO}⁸ Complex that Spontaneously Generates N₂O

One-electron reduction of [Fe­(NO)-(N3PyS)]­BF₄ (<b>1</b>) leads to the production of the metastable nonheme {FeNO}⁸ complex, [Fe­(NO)­(N3PyS)] (<b>3</b>). Complex <b>3</b> is a rare example of a high-spin (<i>S</i> = 1) {FeNO}⁸ and is the first example, to our knowledge, of a mononuclear nonheme {FeNO}⁸ species that generates N₂O. A second, novel route to <b>3</b> involves addition of Piloty’s acid, an HNO donor, to an Fe^II precursor. This work provides possible new insights regarding the mechanism of nitric oxide reductases

UV-visible absorption spectra of the apo- and holo-forms of DH_PDH.

<p>Dotted line, apo-form of DH_PDH; black solid line, holo-form of DH_PDH; blue solid line, reduced form by addition of 1 mM l-fucose. All spectra were recorded in 50 mM HEPES buffer, pH 7.0 at room temperature.</p

Structure of d-glucosone (A) and l-fucose (B) in a ¹C₄ conformation.

<p>Structure of d-glucosone (A) and l-fucose (B) in a ¹C₄ conformation.</p

Multiple alignments of the amino acid sequences of CBM1 of <i>Cc</i>PDH and other known CBM1s.

<p>Residues in bold are highly conserved and those in boxes with a black background are perfect matches. Aromatic residues that are candidates for carbohydrate binding are indicated by a filled arrow, and two pairs of cysteines forming disulfide bonds are indicated by filled and open circles, respectively. <i>Tr</i>CBHI, cellobiohydrolase I (Cel7A) from <i>Trichoderma reesei</i> (accession no. P62694); <i>Pc</i>CBHII, cellobiohydrolase II (Cel6A) from <i>Phanerochaete chrysosporium</i> (Q02321); <i>Pc</i>BGL3A, glucan β-1,3-glucosidase (Bgl) from <i>P</i>. <i>chrysosporium</i> (Q8TGC6); <i>Pc</i>CBHI, cellobiohydrolase I-2 (Cel7D) from <i>P</i>. <i>chrysosporium</i> (Q09431); <i>Tr</i>CBHII, cellobiohydrolase II (Cel6A) from <i>T</i>. <i>reesei</i> (P07987); <i>Pc</i>CBCytb562, carbohydrate-binding cytochrome <i>b</i>₅₆₂ from <i>P</i>. <i>chrysosporium</i> (Q66NB8); <i>Mt</i>CDH, cellobiose dehydrogenase from <i>Myceliophthora thermophila</i> (O74240).</p

Effect of Deglycosylation of Cellobiose Dehydrogenases on the Enhancement of Direct Electron Transfer with Electrodes

Cellobiose dehydrogenase (CDH) is a monomeric extracellular flavocytochrome composed of a catalytic dehydrogenase domain (DH_CDH) containing flavin adenine dinucleotide (FAD), a cytochrome domain (CYT_CDH) containing heme <i>b</i>, and a linker region connecting the two domains. In this work, the effect of deglycosylation on the electrochemical properties of CDH from Phanerochaete chrysosporium (<i>Pc</i>CDH) and Ceriporiopsis subvermispora (<i>Cs</i>CDH) is presented. All the glycosylated and deglycosylated enzymes show direct electron transfer (DET) between the CYT_CDH and the electrode. Graphite electrodes modified with deglycosylated <i>Pc</i>CDH (d<i>Pc</i>CDH) and <i>Cs</i>CDH (d<i>Cs</i>CDH) have a 40–65% higher <i>I</i>_max value in the presence of substrate than electrodes modified with their glycosylated counterparts. <i>Cs</i>CDH trapped under a permselective membrane showed similar changes on gold electrodes protected by a thiol-based self-assembled monolayer (SAM), in contrast to <i>Pc</i>CDH for which deglycosylation did not exhibit any different electrocatalytical response on SAM-modified gold electrodes. Glycosylated <i>Pc</i>CDH was found to have a 30% bigger hydrodynamic radius than d<i>Pc</i>CDH using dynamic light scattering. The basic bioelectrochemistry as well as the bioelectrocatalytic properties are presented

Specificity constant values of <i>Cc</i>PDH for various monosaccharides.

<p>Specificity constant values of <i>Cc</i>PDH for various monosaccharides.</p