Robert L. Osborne

The Mechanism of Oxidative Halophenol Dehalogenation by Amphitrite ornata Dehaloperoxidase Is Initiated by H2O2 Binding and Involves Two Consecutive One-Electron Steps: Role of Ferryl Intermediates

The enzymatic globin, dehaloperoxidase (DHP), from the terebellid polychaete Amphitrite ornata is designed to catalyze the oxidative dehalogenation of halophenol substrates. In this study, the ability of DHP to catalyze this reaction by a mechanism involving two consecutive one-electron steps via the normal order of addition of the oxidant cosubstrate (H(2)O(2)) before organic substrate [2,4,6-trichlorophenol (TCP)] is demonstrated. Specifically, 1 equiv of H(2)O(2) will fully convert 1 equiv of TCP to 2,6-dichloro-1,4-benzoquinone, implicating the role of multiple ferryl [Fe(IV)O] species. A significant amount of heterolytic cleavage of the O-O bond of cumene hydroperoxide, consistent with transient formation of a Compound I [Fe(IV)O/porphyrin pi-cation radical] species, is observed upon its reaction with ferric DHP. In addition, a more stable high-valent Fe(IV)O-containing DHP intermediate [Compound II (Cpd II) or Compound ES] is characterized by UV-visible absorption and magnetic circular dichroism spectroscopy. Spectral similarities are seen between this intermediate and horse heart myoglobin Cpd II. It is also shown in single-turnover experiments that the DHP Fe(IV)O intermediate is an active oxidant in halophenol oxidative dehalogenation. Furthermore, reaction of DHP with 4-chlorophenol leads to a dimeric product. The results presented herein are consistent with a normal peroxidase order of addition of the oxidant cosubstrate (H(2)O(2)) followed by organic substrate (TCP) and indicate that the enzymatic mechanism of DHP-catalyzed oxidative halophenol dehalogenation involves two consecutive one-electron steps with a dissociable radical intermediate.

Probing the oxyferrous and catalytically active ferryl states of Amphitrite ornata dehaloperoxidase by cryoreduction and EPR/ENDOR spectroscopy. Detection of compound I.

Dehaloperoxidase (DHP) from Amphitrite ornata is a heme protein that can function both as a hemoglobin and as a peroxidase. This report describes the use of 77 K cryoreduction EPR/ENDOR techniques to study both functions of DHP. Cryoreduced oxyferrous [Fe(II)-O(2)] DHP exhibits two EPR signals characteristic of a peroxoferric [Fe(III)-O(2)(2-)] heme species, reflecting the presence of conformational substates in the oxyferrous precursor. (1)H ENDOR spectroscopy of the cryogenerated substates shows that H-bonding interactions between His N(ε)H and heme-bound O(2) in these conformers are similar to those in the β-chain of oxyferrous hemoglobin A (HbA) and oxyferrous myoglobin, respectively. Decay of cryogenerated peroxoferric heme DHP intermediates upon annealing at temperatures above 180 K is accompanied by the appearance of a new paramagnetic species with an axial EPR signal with g(⊥) = 3.75 and g(∥) = 1.96, characteristic of an S = 3/2 spin state. This species is assigned to Compound I (Cpd I), in which a porphyrin π-cation radical is ferromagnetically coupled with an S = 1 ferryl [Fe(IV)═O] ion. This species was also trapped by rapid freeze-quench of the ambient-temperature reaction mixture of ferric [Fe(III)] DHP and H(2)O(2). However, in the latter case Cpd I is reduced very rapidly by a nearby tyrosine to form Cpd ES [(Fe(IV)═O)(porphyrin)/Tyr(•)]. Addition of the substrate analogue 2,4,6-trifluorophenol (F(3)PhOH) suppresses formation of the Cpd I intermediate during annealing of cryoreduced oxyferrous DHP at 190 K but has no effect on the spectroscopic properties of the remaining cryoreduced oxyferrous DHP intermediates and kinetics of their decay. These observations indicate that substrate (i) binds to oxyferrous DHP outside of the distal pocket and (ii) can reduce Cpd I to Cpd II [Fe(IV)═O]. These assumptions are also supported by the observation that F(3)PhOH has only a small effect on the EPR properties of radiolytically cryooxidized and cryoreduced ferrous [Fe(II)] DHP. EPR spectra of cryoreduced ferrous DHP disclose the multiconformational nature of the ferrous DHP precursor. The observation and characterization of Cpds I, II, and ES in the absence and in the presence of F(3)PhOH provides definitive evidence of a mechanism involving consecutive one-electron steps and clarifies the role of all intermediates formed during turnover.

Replacement of the axial histidine heme ligand with cysteine in nitrophorin 1: spectroscopic and crystallographic characterization

To evaluate the potential of using heme-containing lipocalin nitrophorin 1 (NP1) as a template for protein engineering, we have replaced the native axial heme-coordinating histidine residue with glycine, alanine, and cysteine. We report here the characterization of the cysteine mutant H60C_NP1 by spectroscopic and crystallographic methods. The UV/vis, resonance Raman, and magnetic circular dichroism spectra suggest weak thiolate coordination of the ferric heme in the H60C_NP1 mutant. Reduction to the ferrous state resulted in loss of cysteine coordination, while addition of exogenous imidazole ligands gave coordination changes that varied with the ligand. Depending on the substitution of the imidazole, we could distinguish three heme coordination states: five-coordinate monoimidazole, six-coordinate bisimidazole, and six-coordinate imidazole/thiolate. Ligand binding affinities were measured and found to be generally 2–3 orders of magnitude lower for the H60C mutant relative to NP1. Two crystal structures of the H60C_NP1 in complex with imidazole and histamine were solved to 1.7- and 1.96-Å resolution, respectively. Both structures show that the H60C mutation is well tolerated by the protein scaffold and suggest that heme–thiolate coordination in H60C_NP1 requires some movement of the heme within its binding cavity. This adjustment may be responsible for the ease with which the engineered heme–thiolate coordination can be displaced by exogenous ligands.

Interdomain long-range electron transfer becomes rate-limiting in the Y216A variant of tyramine β-monooxygenase.

The enzyme tyramine β-monooxygenase (TβM) belongs to a small eukaryotic family of physiologically important mononuclear dicopper monooxygenases. The properties of this family include noncoupled mononuclear copper centers ~11 Å apart, with Cu(M) performing C-H and O(2) activation and Cu(H) functioning as an electron storage site [Klinman, J. P. (2006) J. Biol. Chem. 281, 3013-3016]. A conserved tyrosine (Y216 in TβM) is positioned between the copper domains and is associated with Cu(H) (through an interaction with a Cu(H)-coordinating histidine). Mutations at Y216 (to W, I, and A) indicate little or no difference in electron paramagnetic resonance spectra, while X-ray absorption spectroscopy studies show only a very small decrease in distance between Cu(M) and its Met471 ligand in reduced enzyme. High-performance liquid chromatography assays demonstrate that turnover of substrate is complete with Y216W and Y216I, whereas Y216A undergoes a secondary inactivation that is linked to oxidation of ligands at Cu(M). Steady-state kinetic and isotope effect measurements were investigated. The significantly elevated K(m,Tyr) for Y216A, together with a very large (D)(k(cat)/K(m,Tyr)) of ~12, indicates a major impact on the binding of substrate at the Cu(M) site. The kinetic and isotopic parameters lead to estimated rate constants for C-H bond cleavage, dissociation of substrate from the Cu(M) site, and, in the case of Y216A, the rate of electron transfer (ET) from Cu(H) to Cu(M). These studies uncover a rate-limiting ET within the solvent-filled interface and lead to a paradigm shift in our understanding of the mononuclear dicopper monooxygenases.

Inactivation of Met471Cys Tyramine β-Monooxygenase Results from Site-Specific Cysteic Acid Formation

Tyramine β-monooxygenase (TβM), the insect homologue of dopamine β-monooxygenase, is a neuroregulatory enzyme that catalyzes the β-hydroxylation of tyramine to yield octopamine. Mutation of the methionine (Met) ligand to Cu(M) of TβM, Met471Cys, yielded a form of TβM that is catalytically active but susceptible to inactivation during turnover [Hess, C. R., Wu, Z., Ng, A., Gray, E. E., McGuirl, M. M., and Klinman, J. P. (2008) J. Am. Chem. Soc. 130, 11939-11944]. Further, although the wild-type (WT) enzyme undergoes coordination of Met471 to Cu(M) in its reduced form, the generation of Met471Cys almost completely eliminates this interaction [Hess, C. R., Klinman, J. P., and Blackburn, N. J. (2010) J. Biol. Inorg. Chem. 15, 1195-1207]. The aim of this study is to identify the chemical consequence of the poor ability of Cys to coordinate Cu(M). We show that Met471Cys TβM is ~5-fold more susceptible to inactivation than the WT enzyme in the presence of the cosubstrate/reductant ascorbate and that this process is not facilitated by the substrate tyramine. The resulting 50-fold smaller ratio for turnover to inactivation in the case of Met471Cys prevents full turnover of the substrate under all conditions examined. Liquid chromatography-tandem mass spectrometry analysis of proteolytic digests of inactivated Met471Cys TβM leads to the identification of cysteic acid at position 471. While both Met and Cys side chains are expected to be similarly subject to oxidative damage in proteins, the enhanced reactivity of Met471Cys toward solution oxidants in TβM is attributed to its weaker interaction with Cu(I)(M).