Rieske dearomatizing dioxygenases utilize a Rieske iron-sulfur cluster and a mononuclear Fe(II) located 15 Å across a subunit boundary to catalyze O2-dependent formation of cis-dihydrodiol products from aromatic substrates. During catalysis, O2 binds to the Fe(II) while the substrate bind nearby. Single turnover reactions have shown that one electron from each metal center is required for catalysis. This finding suggested that the reactive intermediate is Fe(III)-(H)peroxo or HO-Fe(V)=O formed by O-O bond scission. Surprisingly, several kinetic phases were observed during the single turnover Rieske cluster oxidation. Here, the Rieske cluster oxidation and product formation steps of a single turnover of benzoate 1,2-dioxygenase are investigated using benzoate and three fluorinated analogs. It is shown that the rate constant for product formation correlates with the reciprocal relaxation time of only the fastest kinetic phase (RRT-1) for each substrate, suggesting that the slower phases are not mechanistically relevant. RRT-1 is strongly dependent on substrate type, suggesting a role for substrate in electron transfer from the Rieske cluster to the mononuclear iron site. This insight, together with the substrate and O2 concentration dependencies of RRT-1, indicates that a reactive species is formed after substrate and O2 binding, but before electron transfer from the Rieske cluster. Computational studies show that RRT-1 is correlated with the electron density at the substrate carbon closest to the Fe(II), consistent with initial electrophilic attack by an Fe(III)-superoxo intermediate. The resulting Fe(III)-peroxo-aryl radical species would then readily accept an electron from the Rieske cluster to complete the cis-dihydroxylation reaction.

Galactose oxidase is a member of a growing class of proteins with novel posttranslationally modified redox-active amino acids (see Figure 1). 1 The unusual nature of these modifications has stimulated interest in the mechanisms by which such cofactors are generated. Recently, the biogenesis of the 2,4,5-trihydroxyphenylalanine quinone (TPQ) cofactor of amine oxidase has been defined. 2 The oxidation of tyrosine to TPQ requires only copper ions and dioxygen, and is not dependent on any accessory proteins. 3 Analogous experiments with galactose oxidase have been hampered by the lack of sufficient quantities of pure precursor (unprocessed, copper-free) protein. Here we report the isolation of an apo, pro-enzyme form of galactose oxidase, and demonstrate that cleavage of the pro-sequence and assembly of the characteristic Tyr • -Cys cofactor are self-processing reactions. Figure 1 illustrates the critical features of the galactose oxidase active site. 4 In the oxidized (active) state tyrosine 272, which is posttranslationally cross-linked to cysteine 228 via a thioether bond, is oxidized to a radical. Thus the [Cu(II) Tyr • -Cys] unit acts as a two-electron acceptor in the oxidation of a wide variety of alcohols to the corresponding aldehydes. The posttranslational cross-link is believed to modulate the reactivity and redox potential of the tyrosyl radical. 5,6 C228 may aid in stabilization of the radical by virtue of the electron-donating properties of the sulfur atom. 5 The oxidized form of galactose oxidase displays a characteristic set of electronic transitions (vida infra) that are also observed in glyoxal oxidase, a galactose oxidase homologue. 7 Heterologous expression 8 of the Fusarium protein in Aspergillus nidulans under copper-limited conditions resulted in the appearance of multiple protein forms ( Figure 2). The molecular weights of the SDS-PAGE bands in Figure 2a, established to be galactose oxidase by Western blotting, 8 were estimated as 70.2, 68.5, and ∼65.5 kDa. N-terminal sequencing established that the fastest migrating protein (lower band, ∼65.5 kDa) corresponds to mature, wild-type galactose oxidase. Mature galactose oxidase migrates on SDS-PAGE with an anomalous molecular weight (65.5 kDa as compared to 68.5 kDa predicted by the sequence), owing to the thioether bond, which produces a stable loop thus preventing full unfolding on treatment with SDS. 8 The middle band ( Figure 2a) has an estimated M r that correlates with the mass of the mature galactose oxidase amino acid sequence, suggesting that it is a form of galactose oxidase lacking the thioether bond. This behavior is mirrored by the variant C228G, which is unable to generate a thioether bond. 8 Finally, the upper band (Figure 2a), having an estimated M r of 70.2 kDa, corresponds to the pro-form with the pro-sequence attached, which was confirmed by the N-terminal sequence data (Table 1). These data suggest that prosequence cleavage and thioether bond formation are separable reactions in vivo.Purification of a homogeneous form of u...

The Rieske dioxygenases are a major subclass of mononuclear nonheme iron enzymes that play an important role in bioremediation. Recently, a high-spin FeIII–(hydro)-peroxy intermediate (BZDOp) has been trapped in the peroxide shunt reaction of benzoate 1,2-dioxygenase. Defining the structure of this intermediate is essential to understanding the reactivity of these enzymes. Nuclear resonance vibrational spectroscopy (NRVS) is a recently developed synchrotron technique that is ideal for obtaining vibrational, and thus structural, information on Fe sites, as it gives complete information on all vibrational normal modes containing Fe displacement. In this study, we present NRVS data on BZDOp and assign its structure using these data coupled to experimentally calibrated density functional theory calculations. From this NRVS structure, we define the mechanism for the peroxide shunt reaction. The relevance of the peroxide shunt to the native FeII/O2 reaction is evaluated. For the native FeII/O2 reaction, an FeIII–superoxo intermediate is found to react directly with substrate. This process, while uphill thermodynamically, is found to be driven by the highly favorable thermodynamics of proton-coupled electron transfer with an electron provided by the Rieske [2Fe-2S] center at a later step in the reaction. These results offer important insight into the relative reactivities of FeIII–superoxo and FeIII–hydroperoxo species in nonheme Fe biochemistry.

The function of the stacking tryptophan, W290, a second coordination sphere residue in galactose oxidase has been investigated via steady-state kinetics measurements, absorption, CD and EPR spectroscopy, and x -ray crystallography of the W290F, W290G, and W290H variants. Enzymatic turnover is significantly lower in the W290 variants. The K m for D-galactose for W290H is similar to wild type, whereas the Km is greatly elevated in W290G and W290F, suggesting a role for W290 in substrate binding/positioning via the -NH group of the indole ring. Hydrogen bonding between W290 and azide in the wild type-azide crystal structure are consistent with this function. W290 modulates the properties and reactivity of the redox-active tyrosine radical; the Y272 tyrosyl radical in both the W290G and W290H variants have elevated redox potentials and are highly unstable compared to the radical in W290F, which has similar properties to the wild type tyrosyl radical. W290 restricts the accessibility of the Y272 radical site to solvent. Crystal structures show that Y272 is significantly more solvent exposed in W290G variant but that W290F limits solvent access comparable to the wild-type indole side chain. Spectroscopic studies indicate that the Cu(II) ground states in the semi-reduced W290 variants are very similar to that of the wild-type protein. In addition, the electronic structures of W290X-azide complexes the variants are also closely similar to the wild type electronic structure. Azide binding and azide-mediated proton uptake by Y495 are perturbed in the variants, indicating that tryptophan also modulates the function of the catalytic base (Y495) in the wild-type enzyme. Thus, W290 plays multiple critical roles in enzyme catalysis, affecting substrate binding, the tyrosyl radical redox potential and stability, and the axial tyrosine function.Over the past twenty years, there has been a growing appreciation for the catalytic utility of protein-derived free radical cofactors in enzymes (1-3). Free radical chemistry is harnessed to catalyze bond activation and molecular rearrangements in a wide variety of enzymes including ribonucleotide reductase (4-7), DNA photolyase (8), cytochrome c peroxidase (9), pyruvateformate lyase (10), lysine-2,3-aminomutase (11), prostaglandin H synthase (12), glyoxal oxidase (13), and galactose oxidase (14).*Authors to whom correspondence should be addressed. Email: dmdooley@montana.edu, Tel: 406-994-4373, FAX: 406 -994-7989; Email: m.j.mcpherson@leeds.ac.uk, Tel: +44 113 233-2595, FAX: +44 113 233-3167. 1 Data deposition: The atomic coordinates and structure factors for W290G, W290F and W290H have been deposited in the Protein Data Bank, www.rcsb.org. † This work was supported by a grant from the National Institutes of Health (GM27659 DMD) and from the Biotechnology and Biological Sciences Research Council (MJM). NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2008 September 9. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptIt...