Redox and Chemical Activities of the Hemes in the Sulfur Oxidation Pathway Enzyme SoxAX

BACKGROUND: SoxAX enzymes initiate microbial oxidation of reduced inorganic sulfur compounds. Their catalytic mechanism is unknown. RESULTS: Cyanide displaces the CysS(-) ligand to the active site heme following reduction by S(2)O(4)(2-) but not Eu(II). CONCLUSION: An active site heme ligand becomes labile on exposure to substrate analogs. SIGNIFICANCE: Elucidation of SoxAX mechanism is necessary to understand a widespread pathway for sulfur compound oxidation. SoxAX enzymes couple disulfide bond formation to the reduction of cytochrome c in the first step of the phylogenetically widespread Sox microbial sulfur oxidation pathway. Rhodovulum sulfidophilum SoxAX contains three hemes. An electrochemical cell compatible with magnetic circular dichroism at near infrared wavelengths has been developed to resolve redox and chemical properties of the SoxAX hemes. In combination with potentiometric titrations monitored by electronic absorbance and EPR, this method defines midpoint potentials (E(m)) at pH 7.0 of approximately +210, -340, and -400 mV for the His/Met, His/Cys(-), and active site His/CysS(-)-ligated heme, respectively. Exposing SoxAX to S(2)O(4)(2-), a substrate analog with E(m) ~-450 mV, but not Eu(II) complexed with diethylene triamine pentaacetic acid (E(m) ~-1140 mV), allows cyanide to displace the cysteine persulfide (CysS(-)) ligand to the active site heme. This provides the first evidence for the dissociation of CysS(-) that has been proposed as a key event in SoxAX catalysis

Synthesis, X-ray Structures, Electronic Properties, and O\u3csub\u3e2\u3c/sub\u3e/NO Reactivities of Thiol Dioxygenase Active-Site Models

Mononuclear non-heme iron complexes that serve as structural and functional mimics of the thiol dioxygenases (TDOs), cysteine dioxygenase (CDO) and cysteamine dioxygenase (ADO), have been prepared and characterized with crystallographic, spectroscopic, kinetic, and computational methods. The high-spin Fe(II) complexes feature the facially coordinating tris(4,5-diphenyl-1-methylimidazol-2-yl)phosphine (Ph2TIP) ligand that replicates the three histidine (3His) triad of the TDO active sites. Further coordination with bidentate l-cysteine ethyl ester (CysOEt) or cysteamine (CysAm) anions yielded five-coordinate (5C) complexes that resemble the substrate-bound forms of CDO and ADO, respectively. Detailed electronic-structure descriptions of the [Fe(Ph2TIP)(LS,N)]BPh4 complexes, where LS,N = CysOEt (1) or CysAm (2), were generated through a combination of spectroscopic techniques [electronic absorption, magnetic circular dichroism (MCD)] and density functional theory (DFT). Complexes 1 and 2 decompose in the presence of O2 to yield the corresponding sulfinic acid (RSO2H) products, thereby emulating the reactivity of the TDO enzymes and related complexes. Rate constants and activation parameters for the dioxygenation reactions were measured and interpreted with the aid of DFT calculations for O2-bound intermediates. Treatment of the TDO models with nitric oxide (NO)—a well-established surrogate of O2—led to a mixture of high-spin and low-spin {FeNO}7 species at low temperature (−70 °C), as indicated by electron paramagnetic resonance (EPR) spectroscopy. At room temperature, these Fe/NO adducts convert to a common species with EPR and infrared (IR) features typical of cationic dinitrosyl iron complexes (DNICs). To complement these results, parallel spectroscopic, computational, and O2/NO reactivity studies were carried out using previously reported TDO models that feature an anionic hydrotris(3-phenyl-5-methyl-pyrazolyl)borate (Ph,MeTp–) ligand. Though the O2 reactivities of the Ph2TIP- and Ph,MeTp-based complexes are quite similar, the supporting ligand perturbs the energies of Fe 3d-based molecular orbitals and modulates Fe–S bond covalency, suggesting possible rationales for the presence of neutral 3His coordination in CDO and ADO

Fast ferrous heme-NO oxidation in nitric oxide synthases.

International audienceDuring catalysis, the heme in nitric oxide synthase (NOS) binds NO before releasing it to the environment. Oxidation of the NOS ferrous heme-NO complex by O2 is key for catalytic cycling, but the mechanism is unclear. We utilized stopped-flow methods to study the reaction of O2 with ferrous heme-NO complexes of inducible and neuronal NOS enzymes. We found that the reaction does not involve heme-NO dissociation, but instead proceeds by a rapid direct reaction of O2 with the ferrous heme-NO complex. This behavior is novel and may distinguish heme-thiolate enzymes, such as NOS, from related heme proteins

Genome sequence of the button mushroom Agaricus bisporus reveals mechanisms governing adaptation to a humic-rich ecological niche

Agaricus bisporus is the model fungus for the adaptation, persistence, and growth in the humic-rich leaf-litter environment. Aside from its ecological role, A. bisporus has been an important component of the human diet for over 200 y and worldwide cultivation of the "button mushroom" forms a multibillion dollar industry. We present two A. bisporus genomes, their gene repertoires and transcript profiles on compost andduringmushroomformation.The genomes encode a full repertoire of polysaccharide-degrading enzymes similar to that of wood-decayers. Comparative transcriptomics of mycelium grown on defined medium, casing-soil, and compost revealed genes encoding enzymes involved in xylan, cellulose, pectin, and protein degradation aremore highly expressed in compost. The striking expansion of heme-thiolate peroxidases and β-etherases is distinctive from Agaricomycotina wood-decayers and suggests a broad attack on decaying lignin and related metabolites found in humic acid-rich environment. Similarly, up-regulation of these genes together with a lignolytic manganese peroxidase, multiple copper radical oxidases, and cytochrome P450s is consistent with challenges posed by complex humic-rich substrates. The gene repertoire and expression of hydrolytic enzymes in A. bisporus is substantially different from the taxonomically related ectomycorrhizal symbiont Laccaria bicolor. A common promoter motif was also identified in genes very highly expressed in humic-rich substrates. These observations reveal genetic and enzymatic mechanisms governing adaptation to the humic-rich ecological niche formed during plant degradation, further defining the critical role such fungi contribute to soil structure and carbon sequestration in terrestrial ecosystems. Genome sequence will expedite mushroom breeding for improved agronomic characteristics

How the Proximal Pocket May Influence the Enantiospecificities of Chloroperoxidase-Catalyzed Epoxidations of Olefins

Chloroperoxidase-catalyzed enantiospecific epoxidations of olefins are of significant biotechnological interest. Typical enantiomeric excesses are in the range of 66%–97% and translate into free energy differences on the order of 1 kcal/mol. These differences are generally attributed to the effect of the distal pocket. In this paper, we show that the influence of the proximal pocket on the electron transfer mechanism in the rate-limiting event may be just as significant for a quantitatively accurate account of the experimentally-measured enantiospecificities

Redox-Dependent Stability, Protonation, and Reactivity of Cysteine-Bound Heme Proteins

Cysteine-bound hemes are key components of many enzymes and biological sensors. Protonation (deprotonation) of the Cys ligand often accompanies redox transformations of these centers. To characterize these phenomena, we have engineered a series of Thr78Cys/Lys79Gly/Met80X mutants of yeast cytochrome c (cyt c) in which Cys78 becomes one of the axial ligands to the heme. At neutral pH, the protonation state of the coordinated Cys differs for the ferric and ferrous heme species, with Cys binding as a thiolate and a thiol, respectively. Analysis of redox-dependent stability and alkaline transitions of these model proteins, as well as comparisons to Cys binding studies with the minimalist heme peptide microperoxidase-8, demonstrate that the protein scaffold and solvent interactions play important roles in stabilizing a particular Cys–heme coordination. The increased stability of ferric thiolate compared with ferrous thiol arises mainly from entropic factors. This robust cyt c model system provides access to all four forms of Cys-bound heme, including the ferric thiol. Protein motions control the rates of heme redox reactions, and these effects are amplified at low pH, where the proteins are less stable. Thermodynamic signatures and redox reactivity of the model Cys-bound hemes highlight the critical role of the protein scaffold and its dynamics in modulating redox-linked transitions between thiols and thiolates

Resonance Raman Spectroscopy Reveals that Substrate Structure Selectively Impacts the Heme-Bound Diatomic Ligands of CYP17

An important function of steroidogenic cytochromes P450 is the transformation of cholesterol to produce androgens, estrogens, and the corticosteroids. The activities of cytochrome P450c17 (CYP17) are essential in sex hormone biosynthesis, with severe developmental defects being a consequence of deficiency or mutations. The first reaction catalyzed by this multifunctional P450 is the 17α-hydroxylation of pregnenolone (PREG) to 17α-hydroxypregnenolone (17-OH PREG) and progesterone (PROG) to 17α-hydroxyprogesterone (17-OH PROG). The hydroxylated products then either are used for production of corticoids or undergo a second CYP17 catalyzed transformation, representing the first committed step of androgen formation. While the hydroxylation reactions are catalyzed by the well-known Compound I intermediate, the lyase reaction is believed to involve nucleophilic attack of the earlier peroxo- intermediate on the C20-carbonyl. Herein, resonance Raman (rR) spectroscopy reveals that substrate structure does not impact heme structure for this set of physiologically important substrates. On the other hand, rR spectra obtained here for the ferrous CO adducts with these four substrates show that substrates do interact differently with the Fe-C-O fragment, with large differences between the spectra obtained for the samples containing 17-OH PROG and 17-OH PREG, the latter providing evidence for the presence of two Fe-C-O conformers. Collectively, these results demonstrate that individual substrates can differentially impact the disposition of a heme-bound ligand, including dioxygen, altering the reactivity patterns in such a way as to promote preferred chemical conversions, thereby avoiding the profound functional consequences of unwanted side reactions

Investigating the Role of the Proximal Cysteine Hydrogen Bonding Network and Distal Pocket in Chloroperoxidase

Chloroperoxidase (CPO) is one of the most versatile heme enzyme isolated from the marine fungus, Caldariomyces fumago. Functionally, CPO can catalyze four types of reactions: peroxidation (peroxidase-like), dismutation (catalase-like), halogenation (halogenase-like), and peroxygenation (P450-like). Structurally, CPO has a distal and proximal pockets that can be best described as a hybrid of classical peroxidase and P450s. As a heme-thiolate protein, CPO contains the conserved proximal Pro28-Cys29-Pro30 stretch found in other members of the family. However, the structural and functional roles of these proline residues remain poorly understood. Site-directed mutagenesis was undertaken to generates three CPO mutants, P28A-, P30A-, P28A/P30A-CPO. The replacement of the rigid proline with a more flexible alanine residue, freed up the back bone amide for the formation of additional amide-sulfur hydrogen bond, allowing the investigation of the importance of these residues in CPO catalysis. The three CPO mutants displayed dramatic difference in ligand binding affinity and catalytic activities relative to WT-CPO. Any mutations on the proline resides within the proximal loop eliminated the halogenation and dismutation activities but enhanced the vii epoxidation and peroxidation activities by 4-14 fold. As the binding affinity for cyanide, the CPO mutants displayed significantly higher dissociation constant relative to WT-CPO. Our results revealed that Pro28 and Pro30 play important roles in maintaining the versatility of CPO. As a versatile enzyme, CPO has great application potential in pharmaceutical and chemical industry due to its ability to catalyze the formation of chiral epoxides. Phe103 and Phe186 located on the distal pocket have been proposed to guard the access of substrates to the ferryl oxygen of the heme center. The interactions of these two phenylalanine residues restricted the size of substrates and regulates CPO’s enantioselectivity. F186A- and F103A/F186A-CPO were generated and characterized where the rate of peroxidation and epoxidation were significantly enhanced at the expense of halogenation and dismutation activities. Our results demonstrated that Phe186 played a subtler role relative to Phe103 in terms of substrate specificity and product enantioselectivity of CPO

Analysis of Heme Iron Coordination in DGCR8: The Heme-Binding Component of the Microprocessor Complex

DGCR8 is the RNA-binding partner of the nuclease Drosha. Their complex (the “Microprocessor”) is essential for processing of long, primary microRNAs (pri-miRNAs) in the nucleus. Binding of heme to DGCR8 is essential for pri-miRNA processing. On the basis of the split Soret ultraviolet–visible (UV–vis) spectrum of ferric DGCR8, bis-thiolate sulfur (cysteinate, Cys–) heme iron coordination of DGCR8 heme iron was proposed. We have characterized DGCR8 heme ligation using the Δ276 DGCR8 variant and combined electron paramagnetic resonance (EPR), magnetic circular dichroism (MCD), electron nuclear double resonance, resonance Raman, and electronic absorption spectroscopy. These studies indicate DGCR8 bis-Cys heme iron ligation, with conversion from bis-thiolate (Cys–/Cys–) axial coordination in ferric DGCR8 to bis-thiol (CysH/CysH) coordination in ferrous DGCR8. Pri-miRNA binding does not perturb ferric DGCR8’s optical spectrum, consistent with the axial ligand environment being separated from the substrate-binding site. UV–vis absorption spectra of the FeII and FeII–CO forms indicate discrete species exhibiting peaks with absorption coefficients substantially larger than those for ferric DGCR8 and that previously reported for a ferrous form of DGCR8. Electron–nuclear double resonance spectroscopy data exclude histidine or water as axial ligands for ferric DGCR8 and favor bis-thiolate coordination in this form. UV–vis MCD and near-infrared MCD provide data consistent with this conclusion. UV–vis MCD data for ferrous DGCR8 reveal features consistent with bis-thiol heme iron coordination, and resonance Raman data for the ferrous–CO form are consistent with a thiol ligand trans to the CO. These studies support retention of DGCR8 cysteine coordination upon reduction, a conclusion distinct from those of previous studies of a different ferrous DGCR8 isoform