Brad S. Pierce

Spectroscopic and computational characterization of substrate-bound mouse cysteine dioxygenase: nature of the ferrous and ferric cysteine adducts and mechanistic implications.

Cysteine dioxygenase (CDO) is a mononuclear non-heme Fe-dependent dioxygenase that catalyzes the initial step of oxidative cysteine catabolism. Its active site consists of an Fe(II) ion ligated by three histidine residues from the protein, an interesting variation on the more common 2-His-1-carboxylate motif found in many other non-heme Fe(II)-dependent enzymes. Multiple structural and kinetic studies of CDO have been carried out recently, resulting in a variety of proposed catalytic mechanisms; however, many open questions remain regarding the structure/function relationships of this vital enzyme. In this study, resting and substrate-bound forms of CDO in the Fe(II) and Fe(III) states, both of which are proposed to have important roles in this enzymes catalytic mechanism, were characterized by utilizing various spectroscopic methods. The nature of the substrate/active site interactions was also explored using the cysteine analogue selenocysteine (Sec). Our electronic absorption, magnetic circular dichroism, and resonance Raman data exhibit features characteristic of direct S (or Se) ligation to both the high-spin Fe(II) and Fe(III) active site ions. The resulting Cys- (or Sec-) bound species were modeled and further characterized using density functional theory computations to generate experimentally validated geometric and electronic structure descriptions. Collectively, our results yield a more complete description of several catalytically relevant species and provide support for a reaction mechanism similar to that established for many structurally related 2-His-1-carboxylate Fe(II)-dependent dioxygenases.

Second-sphere interactions between the C93-Y157 cross-link and the substrate-bound Fe site influence the O2 coupling efficiency in mouse cysteine dioxygenase

Cysteine dioxygenase (CDO) is a non-heme iron enzyme that catalyzes the O₂-dependent oxidation of l-cysteine (l-Cys) to produce cysteinesulfinic acid (CSA). Adjacent to the Fe site of CDO is a covalently cross-linked cysteine-tyrosine pair (C93-Y157). While several theories have been proposed for the function of the C93-Y157 pair, the role of this post-translational modification remains unclear. In this work, the steady-state kinetics and O₂/CSA coupling efficiency were measured for wild-type CDO and selected active site variants (Y157F, C93A, and H155A) to probe the influence of second-sphere enzyme-substrate interactions on catalysis. In these experiments, it was observed that both kcat and the O₂/CSA coupling efficiency were highly sensitive to the presence of the C93-Y157 cross-link and its proximity to the substrate carboxylate group. Complementary electron paramagnetic resonance (EPR) experiments were performed to obtain a more detailed understanding of the second-sphere interactions identified in O₂/CSA coupling experiments. Samples of the catalytically inactive substrate-bound Fe(III)-CDO species were treated with cyanide, resulting in a low-spin (S = ¹/₂) ternary complex. Remarkably, both the presence of the C93-Y157 pair and interactions with the Cys carboxylate group could be readily identified by perturbations to the rhombic EPR signal. Spectroscopically validated active site quantum mechanics/molecular mechanics and density functional theory computational models are provided to suggest a potential role for Y157 in the positioning of the substrate Cys in the active site and to verify the orientation of the g-tensor relative to the CDO Fe site molecular axis.

Synthesis and Characterization of Sterically Encumbered β-Ketoiminate Complexes of Iron(II) and Zinc(II)

The synthesis, structure, and spectroscopic signatures of a series of four-coordinate iron(II) complexes of β-ketoiminates and their zinc(II) analogues are presented. An unusual five-coordinate iron(II) triflate with three oxygen bound protonated β-ketoimines is also synthesized and structurally characterized. Single-crystal X-ray crystallographic analysis reveals that the deprotonated bis(chelate)metal complexes are four-coordinate with various degrees of distortion depending on the degree of steric bulk and the electronics of the metal center. Each of the high-spin iron(II) centers exhibits multiple electronic transitions including ligand π to π*, metal-to-ligand charge transfer, and spin-forbidden d-d bands. The (1)H NMR spectra of the paramagnetic high-spin iron(II) centers are assigned on the basis of chemical shifts, longitudinal relaxation times (T(1)), relative integrations, and substitution of the ligands. The electrochemical studies support variations in the ligand strength. Parallel mode EPR measurements for the isopropyl substituted ligand complex of iron(II) show low-field resonances (g > 9.5) indicative of complex aggregation or crystallite formation. No suitable solvent system or glassing mixture was found to remedy this phenomenon. However, the bulkier diisopropylphenyl substituted ligand exhibits an integer spin signal consistent with an isolated iron(ii) center [S = 2; D = -7.1 ± 0.8 cm(-1); E/D = 0.1]. A tentative molecular orbital diagram is assembled.

Recombinant expression, purification, and characterization of ThmD, the oxidoreductase component of tetrahydrofuran monooxygenase

Tetrahydrofuran monooxygenase (Thm) catalyzes the NADH-and oxygen-dependent hydroxylation of tetrahydrofuran to 2-hydroxytetrahydrofuran. Thm is composed of a hydroxylase enzyme, a regulatory subunit, and an oxidoreductase named ThmD. ThmD was expressed in Escherichia coli as a fusion to maltose-binding protein (MBP) and isolated to homogeneity after removal of the MBP. Purified ThmD contains covalently bound FAD, [2Fe-2S] center, and was shown to use ferricyanide, cytochrome c, 2,6-dichloroindophenol, and to a lesser extent, oxygen as surrogate electron acceptors. ThmD displays 160-fold preference for NADH over NADPH and functions as a monomer. The flavin-binding domain of ThmD (ThmD-FD) was purified and characterized. ThmD-FD displayed similar activity as the full-length ThmD and showed a unique flavin spectrum with a major peak at 463nm and a small peak at 396 nm. Computational modeling and mutagenesis analyses suggest a novel three-dimensional fold or covalent flavin attachment in ThmD.

Steady-state substrate specificity and O2-coupling efficiency of mouse cysteine dioxygenase

Cysteine dioxygenase (CDO) is a non-heme mononuclear iron enzyme that catalyzes the oxygen-dependent oxidation of L-cysteine (Cys) to produce L-cysteine sulfinic acid (CSA). Sequence alignment of mammalian CDO with recently discovered thiol dioxygenase enzymes suggests that the mononuclear iron site within all enzymes in this class share a common 3-His first coordination sphere. This implies a similar mechanistic paradigm among thiol dioxygenase enzymes. Although steady-state studies were first reported for mammalian CDO over 45 years ago, detailed analysis of the specificity for alternative thiol-bearing substrates and their oxidative coupling efficiencies have not been reported for this enzyme. Assuming a similar mechanistic theme among this class of enzymes, characterization of the CDO substrate specificity may provide valuable insight into substrate-active site intermolecular during thiol oxidation. In this work, the substrate-specificity for wild-type Mus musculus CDO was investigated using NMR spectroscopy and LC-MS for a variety of thiol-bearing substrates. Tandem mass spectrometry was used to confirm dioxygenase activity for each non-native substrate investigated. Steady-state Michaelis-Menten parameters for sulfinic acid product formation and O₂-consumption were compared to establish the coupling efficiency for each reaction. In light of these results, the minimal substrate requirements for CDO catalysis and O₂-activation are discussed.

Oxidative Uncoupling in Cysteine Dioxygenase Is Gated by a Proton-Sensitive Intermediate

Cysteine dioxygenase (CDO) is a non-heme mononuclear iron enzyme that catalyzes the O2-dependent oxidation of l-cysteine (Cys) to produce cysteine sulfinic acid (CSA). This enzyme catalyzes the first committed step in Cys catabolism; thus, it is central to mammalian sulfur metabolism and redox homeostasis. Ironically, despite nearly 45 years of continued research on CDO, essentially no information has been reported with respect to its kinetic mechanism. In this work, the timing of chemical steps in the CDO kinetic mechanism is investigated by pH/pD-dependent steady-state kinetics and solvent isotope effects on kcat, kcat/KM, and (O2/CSA) coupling. Normal solvent kinetic isotope effects of 1.45 ± 0.05 and 2.0 ± 0.1 are observed in kcat-pL and kcat/KM-pL profiles, respectively. Proton inventory experiments within the pL-independent region (pL 8.5) suggest multiple solvent-exchangeable protons in flight for both kcat and kcat/KM data. The influence of solvent viscosity was also investigated to probe non-chemical steps and to verify that the apparent isotope effects were not attributable to increased solvent viscosity of D2O reactions relative to H2O. Although solvent viscosity did have a modest influence on kcat and kcat/KM, the response is not sufficient to account for the observed solvent isotope effects. This suggests that product release is only partially rate-limiting for CDO catalysis. Most crucially, proton inventory of (O2/CSA) coupling indicates that a proton-sensitive transition state directly follows O2 activation. Thus, protonation of a transient species preceding Cys oxidation is gated by protons in flight. This behavior provides valuable insight into the kinetically masked transients generated during catalysis.

Structures of proteins of biomedical interest from the Center for Eukaryotic Structural Genomics

The Center for Eukaryotic Structural Genomics (CESG) produces and solves the structures of proteins from eukaryotes. We have developed and operate a pipeline to both solve structures and to test new methodologies. Both NMR and X-ray crystallography methods are used for structure solution. CESG chooses targets based on sequence dissimilarity to known structures, medical relevance, and nominations from members of the scientific community. Many times proteins qualify in more than one of these categories. Here we review some of the structures that have connections to human health and disease.

Peroxide-shunt substrate-specificity for the Salmonella typhimurium O2-dependent tRNA modifying monooxygenase (MiaE).

Post-transcriptional modifications of tRNA are made to structurally diversify tRNA. These modifications alter noncovalent interactions within the ribosomal machinery, resulting in phenotypic changes related to cell metabolism, growth, and virulence. MiaE is a carboxylate bridged, nonheme diiron monooxygenase, which catalyzes the O2-dependent hydroxylation of a hypermodified-tRNA nucleoside at position 37 (2-methylthio-N(6)-isopentenyl-adenosine(37)-tRNA) [designated ms(2)i(6)A37]. In this work, recombinant MiaE was cloned from Salmonella typhimurium , purified to homogeneity, and characterized by UV-visible and dual-mode X-band EPR spectroscopy for comparison to other nonheme diiron enzymes. Additionally, three nucleoside substrate-surrogates (i(6)A, Cl(2)i(6)A, and ms(2)i(6)A) and their corresponding hydroxylated products (io(6)A, Cl(2)io(6)A, and ms(2)io(6)A) were synthesized to investigate the chemo- and stereospecificity of this enzyme. In the absence of the native electron transport chain, the peroxide-shunt was utilized to monitor the rate of substrate hydroxylation. Remarkably, regardless of the substrate (i(6)A, Cl(2)i(6)A, and ms(2)i(6)A) used in peroxide-shunt assays, hydroxylation of the terminal isopentenyl-C4-position was observed with >97% E-stereoselectivity. No other nonspecific hydroxylation products were observed in enzymatic assays. Steady-state kinetic experiments also demonstrate that the initial rate of MiaE hydroxylation is highly influenced by the substituent at the C2-position of the nucleoside base (v0/[E] for ms(2)i(6)A > i(6)A > Cl(2)i(6)A). Indeed, the >3-fold rate enhancement exhibited by MiaE for the hydroxylation of the free ms(2)i(6)A nucleoside relative to i(6)A is consistent with previous whole cell assays reporting the ms(2)io(6)A and io(6)A product distribution within native tRNA-substrates. This observation suggests that the nucleoside C2-substituent is a key point of interaction regulating MiaE substrate specificity.

Autoinduction, purification, and characterization of soluble α-globin chains of crocodile (Crocodylus siamensis) hemoglobin in Escherichia coli.

We have established a method to express soluble heme-bound recombinant crocodile (Crocodylus siamensis) α-globin chain holo-protein in bacteria (Escherichia coli) using an autoinduction system without addition of exogenous heme. This is the first time that heme-bound crocodile α-globin chains have been expressed in bacteria without in vitro heme reconstitution. The observed molecular mass of purified recombinant α-globin is consistent with that calculated from the primary amino acid sequence of native crocodile (C. siamensis) α-globin. Both the monomeric and the dimeric protein configuration formed by intermolecular disulfide bond could be purified as soluble protein. Spectroscopic characterization [UV-visible, circular dichroism (CD), and electron paramagnetic resonance (EPR)] of purified recombinant α-globin demonstrates nearly identical properties as reported for hemoglobin and myoglobin isolated from other organisms. For comparison, cyanide and nitric oxide binding of purified α-globin was also investigated. These results suggested that C. siamensis α-globin expressed in E. coli was folded correctly with proper incorporation of the heme cofactor. The expression method we now describe can facilitate production and isolation of individual globin chains in order to further study the mechanism and assembly of crocodile hemoglobin.

Steady-state kinetics and spectroscopic characterization of enzyme-tRNA interactions for the non-heme diiron tRNA-monooxygenase, MiaE

MiaE [2-methylthio-N(6)-isopentenyl-adenosine(37)-tRNA monooxygenase] isolated from Salmonella typhimurium is a unique non-heme diiron enzyme that catalyzes the O2-dependent post-transcriptional allylic hydroxylation of a hypermodified nucleotide (ms(2)i(6)A37) at position 37 of selected tRNA molecules to produce 2-methylthio-N(6)-(4-hydroxyisopentenyl)-adenosine(37). In this work, isopentenylated tRNA substrates for MiaE were produced from small RNA oligomers corresponding to the anticodon stem loop (ACSL) region of tRNA(Trp) using recombinant MiaA and dimethylallyl pyrophosphate. Steady-state rates for MiaE-catalyzed substrate hydroxylation were determined using recombinant ferredoxin (Fd) and ferredoxin reductase (FdR) to provide a catalytic electron transport chain (ETC) using NADPH as the sole electron source. As with previously reported peroxide-shunt assays, steady-state product formation retains nearly stoichiometric (>98%) E stereoselectivity. MiaE-catalyzed i(6)A-ACSL(Trp) hydroxylation follows Michaelis-Menten saturation kinetics with kcat, KM, and V/K determined to be 0.10 ± 0.01 s(-1), 9.1 ± 1.5 μM, and ∼11000 M(-1) s(-1), respectively. While vastly slower, MiaE-catalyzed hydroxylation of free i(6)A nucleoside could also be observed using the (Fd/FdR)-ETC assay. By comparison to the V/K determined for i(6)A-ACSL substrates, an ∼6000-fold increase in enzymatic efficiency is imparted by ACSL(Trp)-MiaE interactions. The impact of substrate tRNA-MiaE interactions on protein secondary structure and active site electronic configuration was investigated using circular dichroism, dual-mode X-band electron paramagnetic resonance, and Mössbauer spectroscopies. These studies demonstrate that binding of tRNA to MiaE induces a protein conformational change that influences the electronic structure of the diiron site analogous to what has been observed for various bacterial multicomponent diiron monooxygenases upon titration with their corresponding effector proteins. These observations suggest that substrate-enzyme interactions may play a pivotal role in modulating the reactivity of the MiaE diiron active site. Moreover, the simplified monomeric (α) protein configuration exhibited by MiaE provide an unparalleled opportunity to study the impact of protein-effector interactions on non-heme diiron site geometry and reactivity.