Emma J. Murphy

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.

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.

Fascaplysin-inspired diindolyls as selective inhibitors of CDK4/cyclin D1

We present the design, synthesis and biological activity of a new series of substituted 3-(2-(1H-indol-1-yl)ethyl)-1H-indoles and 1,2-di(1H-indol-1-yl)alkanes as selective inhibitors of CDK4/cyclin D1. The compounds were designed to explore the relationship between the connection mode of the indolyl moieties and their CDK inhibitory activities. We found all the above-mentioned designed compounds to be selective inhibitors of CDK4/cyclin D1 compared to the closely related CDK2/cyclin A, with IC(50) for the best compounds 10m and 13a being 39 and 37microm, respectively.

New insights into the mechanism of odorant detection by the malaria - transmitting mosquito anopheles gambiae

Anopheles gambiae mosquitoes that transmit Plasmodium falciparum malaria use a series of olfactory cues present in human sweat to locate their hosts for a blood meal. Recognition of these odor cues occurs through the interplay of odorant receptors and odorant-binding proteins (OBPs) that bind to odorant molecules and transport and present them to the receptors. Recent studies have implicated potential heterodimeric interactions between two OBPs, OBP1 and OBP4, as important for perception of indole by the mosquito (Biessmann, H., Andronopoulou, E., Biessmann, M. R., Douris, V., Dimitratos, S. D., Eliopoulos, E., Guerin, P. M., Iatrou, K., Justice, R. W., Kröber, T., Marinotti, O., Tsitoura, P., Woods, D. F., and Walter, M. F. (2010) PLoS ONE 5, e9471; Qiao, H., He, X., Schymura, D., Ban, L., Field, L., Dani, F. R., Michelucci, E., Caputo, B., della Torre, A., Iatrou, K., Zhou, J. J., Krieger, J., and Pelosi, P. (2011) Cell. Mol. Life Sci. 68, 1799–1813). Here we present the 2.0 Å crystal structure of the OBP4-indole complex, which adopts a classical odorant-binding protein fold, with indole bound at one end of a central hydrophobic cavity. Solution-based NMR studies reveal that OBP4 exists in a molten globule state and binding of indole induces a dramatic conformational shift to a well ordered structure, and this leads to the formation of the binding site for OBP1. Analysis of the OBP4-OBP1 interaction reveals a network of contacts between residues in the OBP1 binding site and the core of the protein and suggests how the interaction of the two proteins can alter the binding affinity for ligands. These studies provide evidence that conformational ordering plays a key role in regulating heteromeric interactions between OBPs.

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.

CO Binding and Ligand Discrimination in Human Myeloperoxidase

Despite the fact that ferrous myeloperoxidase (MPO) can bind both O(2) and NO, its ability to bind CO has been questioned. UV/visible spectroscopy was used to confirm that CO induces small spectral shifts in ferrous MPO, and Fourier transform infrared difference spectroscopy showed definitively that these arose from formation of a heme ferrous-CO compound. Recombination rates after CO photolysis were monitored at 618 and 645 nm as a function of CO concentration and pH. At pH 6.3, k(on) and k(off) were 0.14 mM(-1) x s(-1) and 0.23 s(-1), respectively, yielding an unusually high K(D) of 1.6 mM. This affinity of MPO for CO is 10 times weaker than its affinity for O(2). The observed rate constant for CO binding increased with increasing pH and was governed by a single protonatable group with a pK(a) of 7.8. Fourier transform infrared spectroscopy revealed two different conformations of bound CO with frequencies at 1927 and 1942 cm(-1). Their recombination rate constants were identical, indicative of two forms of bound CO that are in rapid thermal equilibrium rather than two distinct protein populations with different binding sites. The ratio of bound states was pH-dependent (pK(a) approximately 7.4) with the 1927 cm(-1) form favored at high pH. Structural factors that account for the ligand-binding properties of MPO are identified by comparisons with published data on a range of other ligand-binding heme proteins, and support is given to the recent suggestion that the proximal His336 in MPO is in a true imidazolate state.

Heme enzymes. 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.