The α-keto acid dependent dioxygenases are a major subgroup within the O2-activating mononuclear non-heme iron enzymes. For these enzymes, the resting ferrous, the substrate plus cofactor-bound ferrous, and the FeIV=O states of the reaction have been well studied. The initial O2-binding and activation steps are experimentally inaccessible and thus are not well understood. In this study, NO is used as an O2 analog to probe the effects of α-keto acid binding in 4-hydroxyphenylpyruvate dioxygenase (HPPD). A combination of EPR, UV-vis absorption, magnetic circular dichroism (MCD), and variable-temperature, variable-field (VTVH) MCD spectroscopies in conjunction with computational models is used to explore the HPPD-NO and HPPD-HPP-NO complexes. New spectroscopic features are present in the α-keto acid bound {FeNO}7 site that reflect the strong donor interaction of the α-keto acid with the Fe. This promotes the transfer of charge from the Fe to NO. The calculations are extended to the O2 reaction coordinate where the strong donation associated with the bound α-keto acid promotes formation of a new, S=1 bridged FeIV-peroxy species. These studies provide insight into the effects of a strong donor ligand on O2 binding and activation by FeII in the α-keto acid dependent dioxygenases and are likely relevant to other subgroups of the O2 activating non-heme ferrous enzymes.

The oxygen activating mononuclear non-heme ferrous enzymes catalyze a diverse range of chemistry yet typically maintain a common structural motif: two histidines and a carboxylate coordinating the iron center in a facial triad. A new Fe II coordinating triad has been observed in two enzymes, diketone cleaving dioxygenase, Dke1, and cysteine dioxygenase (CDO) and is composed of three histidine residues. The effect of this 3 His motif in Dke1 on the geometric and electronic structure of the Fe II center is explored via a combination of absorption, CD, MCD and VTVH MCD spectroscopies and DFT calculations. This geometric and electronic structure of the 3 His triad is compared to that of the classical (2-His-1-carboxylate) facial triad in the α-ketoglutarate (αKG) dependent dioxygenases clavaminate synthase 2 (CS2) and hydroxyphenylpyruvate dioxygenase (HPPD). Comparison of the ligand fields at the Fe II shows little difference between the 3 His and 2-His-1-carboxylate facial triad sites. Acac, the substrate for Dke1, will also bind to HPPD and is determined to be a strong donor, similar to αKG. The major difference between the 3 His and 2-His-1-carboxylate facial triad sites is in MLCT transitions observed for both types of triads and reflects their difference in charge. These studies provide insight into the effects of perturbation of the facial triad ligation of the non-heme ferrous enzymes on their geometric and electronic structure and their possible contributions to reactivity.Mononuclear nonheme iron enzymes (MNHFe's) are an important class that are involved with a wide range of medical, pharmaceutical, and environmental applications. They catalyze a diverse range of chemical reactions, most commonly hydroxylation, but also oxidative ring closure, desaturation, carbon-carbon bond and aromatic ring cleavage, hydrogen atom abstraction and halogenation.(1,3) The oxygen activating enzymes -that include the extradiol dioxygenases, pterin-dependent dioxygenases, Rieske dioxygenases and α-keto acid dependent dioxygenases -use Fe II to activate O 2 for attack on the substrate.(1) They usually share a common structural motif for Fe II coordination: a protein-derived facial triad composed of two histidine residues and one carboxylate moiety (hereafter * To whom correspondence should be addressed: Edward I. Solomon Edward.solomon@stanford.edu; Grit D. Straganz: grit.straganz@tugraz.at,. Supporting Information Available Full reference for reference 24; VTVH MCD isotherms for Dke1-acac at 11,100 cm −1 and HPPD-acac at 12,300 cm −1 ; CD and MCD of resting Fe II -HPPD; HOMO and LUMO of free acac; TD-DFT predicted spectra for acac-bound complexes; structures used to calculate water affinity. This material is available free of charge via the Internet at http://pubs.acs.org. referred to as the facial triad).(4,5) Additionally, along with the substrate, most of these enzymes require a cofactor to deliver the reducing equivalents necessary for reactivity. NIH Public AccessRecently, a few exceptions to this facial triad mo...

The O2 activating mononuclear non-heme iron enzymes generally have a common facial triad (2 histidine and one carboxylate (Asp or Glu) residue) ligating FeII at the active site. Exceptions to this motif have recently been identified in non-heme enzymes, including a 3His triad in the diketone cleaving dioxygenase Dke1. This enzyme is used to explore the role of the facial triad in directing reactivity. A combination of spectroscopic studies (UV-vis absorption, MCD, and resonance Raman) and DFT calculations is used to define the nature of the binding of the α-keto acid, 4-hydroxyphenlpyruvate (HPP), to the active site in Dke1 and the origin of the atypical cleavage (C2–C3 instead of C1–C2) pattern exhibited by this enzyme in the reaction of α-keto acids with dioxygen. The reduced charge of the 3His triad induces α-keto acid binding as the enolate dianion, rather than the keto monoanion, found for α-keto acid binding to the 2His/1 carboxylate facial triad enzymes. The mechanistic insight from the reactivity of Dke1 with the α-keto acid substrate is then extended to understand the reaction mechanism of this enzyme with its native substrate, acac. This study defines a key role for the 2His/1 carboxylate facial triad in α-keto acid dependent mononuclear non-heme iron enzymes in stabilizing the bound α-keto acid as a monoanion for its decarboxylation to provide the two additional electrons required for O2 activation.

Diketone cleaving enzyme (Dke1) is a dioxygenase with an atypical, 3 histidine ligated, mononuclear non-heme Fe2+ center. To assess the role in enzyme catalysis of the hydrophilic residues in the active site pocket, residues Glu98, Arg80, Tyr70 and Thr107 were subjected to mutational analysis. Steady state and pre-steady state kinetics indicated a role for Glu98 in promoting both substrate binding and O2 reduction. Additionally, the Glu98-substitution eliminated the pH dependence of substrate binding (kcatapp/KMapp-pH profile) present in wild type Dke1 (pKa 6.3±0.4 and 8.4±0.4). MCD spectroscopy revealed that the Glu98→Gln mutation leads to the conversion of the six-coordinate (6C) resting Fe2+ center present in the wild type enzyme at pH 7.0 to a mixture of 5C and 6C sites. The 6C geometry was restored with a pH-shift to 9.5 which also resulted in ligand field (LF) energy splittings identical to that found for wild type (WT) Dke1 at pH 9.5. In WT Dke1 these LF transitions are shifted up in energy by ~300 cm-1 at pH 9.5 relative to pH 7.0. These data, combined with CD pH titrations which reveal a pKa of ~8.2 for resting WT Dke1 and the Glu98→Gln variant, indicate the deprotonation of a metal ligated water. Together, the kinetic and spectroscopic data reveal a stabilizing effect of Glu98 on the 6C geometry of the metal center, priming it for substrate ligation. Arg80 and Tyr70 are shown to promote O2 reduction, while Thr107 stabilizes the Fe(II) cofactor.

DNA Protection during Starvation (Dps) proteins are mini-ferritins found in bacteria and archaea that provide protection from uncontrolled Fe(II)/O radical chemistry; thus the catalytic sites are targets for antibiotics against pathogens, such as anthrax. Ferritin protein cages synthesize ferric oxymineral from Fe(II) and O 2 /H 2 O 2 , which accumulates in the large central cavity; for Dps, H 2 O 2 , is the more common Fe(II) oxidant contrasting with eukaryotic maxi-ferritins that often prefer dioxygen. To better understand the differences in the catalytic sites of maxi versus miniferritins, we used a combination of NIR circular dichroism (CD), magnetic circular dichroism (MCD), and variable-temperature, variable-field MCD (VTVH MCD) to study Fe(II) binding to the catalytic sites of the two B. anthracis mini-ferritins; one in which two Fe(II) react with O 2 exclusively (Dps1) and a second in which both O 2 or H 2 O 2 can react with two Fe(II) (Dps2). Both result in the formation of iron oxy-biomineral. The data show: a single 5 or 6-coordinate Fe(II) in the absence of oxidant; Fe(II) binding to Dps2 is 30 × more stable than Dps1; and the lower limit of K d for binding a second Fe(II), in the absence of oxidant, is 2-3 orders of magnitude weaker than for the binding of the single Fe(II). The data fit an equilibrium model where binding of oxidant facilitates formation of the catalytic site, in sharp contrast to eukaryotic M-ferritins where the binuclear Fe(II) centers are preformed before binding of O 2 . The two different binding sequences illustrate the mechanistic range possible for catalytic sites of the family of ferritins.DNA Protection during Starvation (Dps) proteins, also known as mini-ferritins, are 12-monomer spherical proteins capable of storing iron mineral and thus are part of the ferritin super-family (1-4). Unlike maxi (24-monomer) ferritins, Dps proteins have been shown to bind and protect DNA from oxidation (2-4). The paired Dps proteins in Bacillus anthracis * To whom correspondence should be addressed: Elizabeth C. Theil etheil@chori.org; and Edward I. Solomon, edward.solomon@stanford.edu;. Supporting Information Available CD spectra showing effect of temperature on Dps1 transitions; MCD spectra of Dps1 overlaid with Fe(II) control spectrum, and MCD spectrum with control subtracted; Apparent initial rates of Fe 2+ oxidation in Dps1 & 2 scaled to overlay with the calculated concentration of binuclear Fe(II) active sites occupied at a given Fe 2+ /Dps2 ratio using fixed K D1 and varied K D2 values. This material is available free of charge via the Internet at http://pubs.acs.org. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2011 December 14. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript (Dps1 and Dps2), which share ~60% sequence homology(3), confer greatest protection when both are present. Dps proteins in pathogens, such as those from B. anthracis, are of particular interest as better knowledge of how these proteins p...

Isopenicillin N synthase (IPNS) can have both oxidase and oxygenase activity depending on the substrate. For the native substrate, ACV, oxidase activity occurs; however for the substrate analogue ACOV, which lacks an amide nitrogen, IPNS shows oxygenase activity. The potential energy surfaces for the O-O bond elongation and cleavage were calculated for three different reactions: homolytic cleavage via traditional Fenton chemistry, heterolytic cleavage, and nucleophilic attack. These surfaces show that the hydroperoxide-Ferrous intermediate, formed by O 2 activated H atom abstraction from substrate, can undergo different reaction pathways and that interactions with the substrate govern the pathway. The hydroperoxide hydrogen bonds to the amide nitrogen of ACV polarizing the σ* orbital of the peroxide toward the proximal oxygen, facilitating heterolytic cleavage. For the substrate analogue ACOV, this hydrogen bond is no longer present, leading to nucleophilic attack on the substrate intermediate C-S bond. After cleavage of the hydroperoxide, the two reaction pathways proceed with minimal barriers to result in the closure of the β-lactam ring for the oxidase activity (ACV) or formation of the thiocarboxylate for oxygenase activity (ACOV).Isopenicillin N-synthase is a mononuclear non-heme iron enzyme found in fungi and bacteria that catalyzes the formation of isopenicillin N, a bicyclic precursor to the β lactam antibiotics including the penicillins and cephalosporins. (1,2) IPNS binds a tripeptide substrate δ-(L-aaminoadipoyl)-L-cysteinyl-D-valine (ACV) and performs a four electron oxidative double ring closure, fully reducing one equivalent of O 2 to H 2 O and closing the β lactam and thiazolidine rings of isopenicillin N. (3-6) (Scheme 1) This oxidase reactivity is unusual as most non-heme iron enzymes catalyze oxygenation reactions. Previous studies of the IPNS-ACV {FeNO} 7 complex revealed that a major factor contributing to the oxidase reactivity of IPNS is charge donation from the ACV thiolate ligand, which renders the formation of the Fe III -superoxide complex energetically favorable and drives the reaction only at the Fe center. (7) This single center, one electron reaction allows IPNS to avoid the bridged binding of O 2 between the Fe II and the substrate/cofactor required for its two electron reduction; a reaction generally invoked for the non-heme Fe enzymes, that leads to oxygen insertion. The thiolate coordination of the IPNS ACV substrate further activates the reactive Fe III -superoxide complex of the enzyme through a configuration interaction with the bound superoxide π* orbital which creates * To whom correspondence should be addressed: edward.solomon@stanford.edu; phone: 650-723-4694; fax: 650-725-0259.The reorientation of the hydroperoxide was accomplished through contraction of the distance between the ACV amide nitrogen and cysteinyl carbon to a distance of approximately 2.8Å. (15)(16)(17)(18)(19) In one such analogue ACOV, the amide nitrogen of the ACV valine is replaced with an oxyg...

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