Clive L. Metcalfe

Kinetically inert transition metal complexes that reversibly bind to DNA

Transition metal complexes that reversibly bind to DNA have been studied for almost 30 years. In the last few years a variety of new systems have been developed, employing a range of metal ions and ligand architectures. In many cases, high affinity binding and specific selectivities have been observed. These complexes display properties that make them attractive as probes of DNA structure and function, suggesting that they may find a rôle as prototypical tools for a spectrum of applications, from basic molecular biology to medicine. This review presents an overview of some of the structures and properties of such complexes.

The Tuberculosis Prodrug Isoniazid Bound to Activating Peroxidases.

Isoniazid (INH, isonicotinic acid hydrazine) is one of only two therapeutic agents effective in treating tuberculosis. This prodrug is activated by the heme enzyme catalase peroxidase (KatG) endogenous to Mycobacterium tuberculosis but the mechanism of activation is poorly understood, in part because the binding interaction has not been properly established. The class I peroxidases ascorbate peroxidase (APX) and cytochrome c peroxidase (CcP) have active site structures very similar to KatG and are also capable of activating isoniazid. We report here the first crystal structures of complexes of isoniazid bound to APX and CcP. These are the first structures of isoniazid bound to any activating enzymes. The structures show that isoniazid binds close to the δ-heme edge in both APX and CcP, although the precise binding orientation varies slightly in the two cases. A second binding site for INH is found in APX at the γ-heme edge close to the established ascorbate binding site, indicating that the γ-heme edge can also support the binding of aromatic substrates. We also show that in an active site mutant of soybean APX (W41A) INH can bind directly to the heme iron to become an inhibitor and in a different mode when the distal histidine is replaced by alanine (H42A). These structures provide the first unambiguous evidence for the location of the isoniazid binding site in the class I peroxidases and provide rationalization of isoniazid resistance in naturally occurring KatG mutant strains of M. tuberculosis.

Neutron cryo-crystallography captures the protonation state of ferryl heme in a peroxidase

Peroxidase proton placement Heme enzymes catalyze a variety of biochemical oxidations through the activation of oxygen by iron. Casadei et al. used neutron crystallography to elucidate the mechanism of cytochrome c peroxidase (see the perspective by Groves and Boaz). In the highly reactive intermediate state termed compound I, the iron(IV) oxo, or ferryl, fragment was not protonated, whereas a nearby histidine residue was protonated. The sensitivity of neutron scattering to proton locations revealed these protonation states, where more common techniques, such as x-ray diffraction, have yielded more ambiguous results. Science, this issue p. 193; see also p. 142 The sensitivity of neutron scattering to proton locations clarifies the acid/base chemistry of a biochemical oxidation. [Also see Perspective by Groves and Boaz] Heme enzymes activate oxygen through formation of transient iron-oxo (ferryl) intermediates of the heme iron. A long-standing question has been the nature of the iron-oxygen bond and, in particular, the protonation state. We present neutron structures of the ferric derivative of cytochrome c peroxidase and its ferryl intermediate; these allow direct visualization of protonation states. We demonstrate that the ferryl heme is an Fe(IV)=O species and is not protonated. Comparison of the structures shows that the distal histidine becomes protonated on formation of the ferryl intermediate, which has implications for the understanding of O–O bond cleavage in heme enzymes. The structures highlight the advantages of neutron cryo-crystallography in probing reaction mechanisms and visualizing protonation states in enzyme intermediates.

Nature of the ferryl heme in Compounds I and II.

Heme enzymes are ubiquitous in biology and catalyze a vast array of biological redox processes. The formation of high valent ferryl intermediates of the heme iron (known as Compounds I and Compound II) is implicated for a number of catalytic heme enzymes, but these species are formed only transiently and thus have proved somewhat elusive. In consequence, there has been conflicting evidence as to the nature of these ferryl intermediates in a number of different heme enzymes, in particular the precise nature of the bond between the heme iron and the bound oxygen atom. In this work, we present high resolution crystal structures of both Compound I and Compound II intermediates in two different heme peroxidase enzymes, cytochrome c peroxidase and ascorbate peroxidase, allowing direct and accurate comparison of the bonding interactions in the different intermediates. A consistent picture emerges across all structures, showing lengthening of the ferryl oxygen bond (and presumed protonation) on reduction of Compound I to Compound II. These data clarify long standing inconsistencies on the nature of the ferryl heme species in these intermediates.

An analysis of substrate binding interactions in the heme peroxidase enzymes: A structural perspective

The interactions of heme peroxidase enzymes with their substrates have been studied for many years, but only in the last decade or so has structural information begun to appear. This review looks at crystal structures for a number of heme peroxidases in complex with a number of (mainly organic) substrates. It examines the nature and location of the binding interaction, and explores functional similarities and differences across the family.

Proton Delivery to Ferryl Heme in a Heme Peroxidase: Enzymatic Use of the Grotthuss Mechanism

We test the hypothesized pathway by which protons are passed from the substrate, ascorbate, to the ferryl oxygen in the heme enzyme ascorbate peroxidase (APX). The role of amino acid side chains and bound solvent is demonstrated. We investigated solvent kinetic isotope effects (SKIE) for the wild-type enzyme and several site-directed replacements of the key residues which form the proposed proton path. Kinetic constants for H(2)O(2)-dependent enzyme oxidation to Compound I, k(1), and subsequent reduction of Compound II, k(3), were determined in steady-state assays by variation of both H(2)O(2) and ascorbate concentrations. A high value of the SKIE for wild type APX ((D)k(3) = 4.9) as well as a clear nonlinear dependence on the deuterium composition of the solvent in proton inventory experiments suggest the simultaneous participation of several protons in the transition state for proton transfer. The full SKIE and the proton inventory data were modeled by applying Gross-Butler-Swain-Kresge theory to a proton path inferred from the known structure of APX. The model has been tested by constructing and determining the X-ray structures of the R38K and R38A variants and accounts for their observed SKIEs. This work confirms APX uses two arginine residues in the proton path. Thus, Arg38 and Arg172 have dual roles, both in the formation of the ferryl species and binding of ascorbate respectively and to facilitate proton transfer between the two.

Crystal Structure of Guaiacol and Phenol Bound to a Heme Peroxidase.

Guaiacol is a universal substrate for all peroxidases, and its use in a simple colorimetric assay has wide applications. However, its exact binding location has never been defined. Here we report the crystal structures of guaiacol bound to cytochrome c peroxidase (CcP). A related structure with phenol bound is also presented. The CcP–guaiacol and CcP–phenol crystal structures show that both guaiacol and phenol bind at sites distinct from the cytochrome c binding site and from the δ‐heme edge, which is known to be the binding site for other substrates. Although neither guaiacol nor phenol is seen bound at the δ‐heme edge in the crystal structures, inhibition data and mutagenesis strongly suggest that the catalytic binding site for aromatic compounds is the δ‐heme edge in CcP. The functional implications of these observations are discussed in terms of our existing understanding of substrate binding in peroxidases [Gumiero A et al. (2010) Arch Biochem Biophys500, 13–20].

Peroxide-dependent formation of a covalent link between Trp51 and the heme in cytochrome c peroxidase.

Ascorbate peroxidase (APX), cytochrome c peroxidase (CcP), and the catalase-peroxidases (KatG) share very similar active site structures and are distinguished from other peroxidases by the presence of a distal tryptophan residue. In KatG, this distal tryptophan forms a covalent link to an adjacent tyrosine residue, which in turn links to a methionine residue. We have previously shown [ Pipirou, Z. et al. ( 2007 ) Biochemistry 46 , 2174 - 2180 ] that reaction of APX with peroxide leads, over long time scales, to formation of a covalent link with the distal tryptophan (Trp41) in a mechanism that proceeds through initial formation of a compound I species bearing a porphyrin pi-cation radical followed by radical formation on Trp41, as implicated in the KatG enzymes. Formation of such a covalent link in CcP has never been reported, and we proposed that this could be because compound I in CcP uses Trp191 instead of a porphyrin pi-cation radical. To test this, we have examined the reactivity of the W191F variant of CcP with H(2)O(2), in which formation of a porphyrin pi-cation radical occurs. We show, using electronic spectroscopy, HPLC, and mass spectroscopy, that in W191F partial formation of a covalent link from Trp51 to the heme is observed, as in APX. Radical formation on Trp51, as seen for KatG and APX, is implicated; this is supported by QM/MM calculations. Collectively, the data show that all three members of the class I heme peroxidases can support radical formation on the distal tryptophan and that the reactivity of this radical can be controlled either by the protein structure or by the nature of the compound I intermediate.

Engineering the Substrate Specificity and Reactivity of a Heme Protein: Creation of an Ascorbate Binding Site in Cytochrome c Peroxidase†

The binding of substrates to heme enzymes has been widely assumed to occur at the so-called delta-heme edge. Recently, however, a number of examples have appeared in which substrate binding at an alternative site, the gamma-heme edge, is also possible. In previous work [Sharp et al. (2003) Nat. Struct. Biol. 10, 303-307], we showed that binding of ascorbate to ascorbate peroxidase occurred at the gamma-heme edge. Here, we show that the closely related cytochrome c peroxidase enzyme can duplicate the substrate binding properties of ascorbate peroxidase through the introduction of relatively modest structural changes at Tyr36 and Asn184. Hence, crystallographic data for the Y36A/N184R/W191F triple variant of cytochrome c peroxidase shows ascorbate bound to the gamma-heme edge, with hydrogen bonds to the heme propionate and Arg184. In parallel mechanistic studies in variants incorporating the W191F mutation, we show that a transient porphyrin pi-cation radical in Compound I of cytochrome c peroxidase, analogous to that observed in ascorbate peroxidase, is competent for ascorbate oxidation but that under steady state conditions this intermediate decays too rapidly to sustain efficient turnover of ascorbate. The results are discussed in terms of our more general understanding of substrate oxidation across other heme proteins, and the emerging role of the heme propionates at the gamma-heme edge.

A ruthenium dipyridophenazine complex that binds preferentially to GC sequences

Uniquely, a Ru11 complex of the dppz ligand shows a preference for GC sequences of DNA.