Electron factors in the properties of cytochrome P-450 and mechanism of activation of molecular oxygen

A quantum-chemical calculation was carried out for the electronic structures of coordination compounds of general formula: FeP(L1)(L2) (P--porphin; L1 = SHCH3, [SCH3]-, [SC6F4H]-; L2 = CO, NO, O2), modeling the active site of cytochrome P450. It was shown that Coulomb repulsion between the electrons of the sulfur lone pair leads to the transfer of the electronic density from the ligands L1 = [SCH3]- or [SC6F4H]- to the porphyrin of/and to the L2 ligand. This explains the origin of the band at 450 nm in the absorption spectra of the complexes of cytochrome P450 with CO, the absence of such a band in those with O2, and the strong activation of dioxygen by cytochrome P450.     

Theoretical study of model compound I complexes of horseradish peroxidase and catalase.

Theoretical studies of the electronic structure and spectra of models for the ferric resting state and Compound I intermediates of horseradish peroxidase (HRP-I) and catalase (CAT-I) have been performed using the INDO-RHF/CI method. The goals of these studies were twofold: i) to determine whether the axial ligand of HRP is best described as imidazole or imidazolate, and ii) to address the long-standing question of whether HRP-I and CAT-I are a1u and a2u tau cation radicals. Only the imidazolate HRP-I model led to a calculated electronic spectra consistent with the experimentally observed significant reduction in the intensity of the Soret band compared with the ferric resting state. These results provide compelling evidence for significant proton transfer to the conserved Asp residue by the proximal histidine. The origin of the observed reduction of the Soret band intensity in HRP-I and CAT-I spectra has been examined and found to be caused by the mixing of charge transfer transitions into the predominantly porphyrin tau-tau transitions. For both HRP-I and CAT-I, the a1u porphyrin tau cation state is the lowest energy, and it is further stabilized by both the anionic form of the ligand and the porphyrin ring substituents of protoporphyrin-IX. The calculated values of quadrupole-splitting observed in the Mossbauer resonance of HRP-I and CAT-I are similar for the a1u and a2u tau cation radicals. Electronic spectrum of the a1u tau cation radical of HRP-I are more similar to the observed spectra, whereas the spectra of both a1u tau and a2u tau cation radicals of CAT-I resemble the observed spectra. These results also indicate the limitations of using any one observable property to try to distinguish between these states. Taken together, comparison of calculated and observed properties indicate that there is no compelling reason to invoke the higher energy a2u tau cation radical as the favored state in HRP-I and CAT-I. Both ground-state properties and electronic spectra are consistent with the a1u tau cation radical.     

Calorimetric studies of oxyhemoglobin dissociation. II. Erythrocytic oxygen depletion by sodium dithionite

Dithionite causes the depletion of dioxygen from suspensions of erythrocytes by reduction of the external dioxygen and not by diffusion into the cell. The molar enthalpy for the reduction shows a small difference with respect to the values found for free hemoglobin; and the normal stoichiometry of 2 moles dithionite/mole dioxygen found there is not observed with erythrocytes. At low hematocrit, the stoichiometry is 2.6:1 and decreases to 1.5:1 at high hematocrit. The change is not due to differences in the hemoglobin saturation or to an inability of dithionite to reduce all dioxygen present at the higher hematocrit. Neither catalase nor peroxidase added to the extracellular volume significantly alters the stoichiometry or the enthalpy of dioxygen reduction by dithionite. Addition of superoxide dismutase, however, restores the normal stoichiometry at high hematocrit and further increases the stoichiometry at low hematocrit. The calorimetrical signal of hydrogen peroxide, clearly seen with free dioxygen, is not present with erythrocytes. In all these cases the total heat evolved is the same.     

A novel diiron complex as a functional model for hemerythrin

Diiron(II) complexes with a novel dinucleating polypyridine ligand, N,N,N',N'-tetrakis(6-pivalamido-2-pyridylmethyl)-1,3-diaminopropan-2-ol (HTPPDO), were synthesized as functional models of hemerythrin. Structural characterization of the complexes, [Fe2II(Htppdo)(PhCOO)](ClO4)3 (1), [Fe2II(Htppdo)((p-Cl)PhCOO)](ClO4)3 (2), [Fe2II(Htppdo)((p-Cl)PhCOO)](BF4)3 (2') and [Fe2II(tppdo)((p-Cl)PhCOO)](ClO4)2 (3), were accomplished by electronic absorption, and IR spectroscopic, electrochemical, and X-ray diffraction methods. The crystal structures of 1 and 2' revealed that the two iron atoms are asymmetrically coordinated with HTPPDO and bridging benzoate. One of the iron centers (Fe(1)) has a seven-coordinate capped octahedral geometry comprised of an N3O4 donor set which includes the propanol oxygen of HTPPDO. The other iron center (Fe(2)) forms an octahedron with an N3O3 donor set and one vacant site. The two iron atoms are bridged by benzoate (1) or p-chlorobenzoate (2). On the other hand, both Fe atoms of complex 3 are both symmetrically coordinated with N3O4 donors and two bridging ligands; benzoate and the propanolate of TPPDO. Reactions of these complexes with dioxygen were followed by electronic absorption, resonance Raman and ESR spectroscopies. Reversible dioxygen-binding was demonstrated by observation of an intense LMCT band for O2(2-) to Fe(III) at 610 (1) and 606 nm (2) upon exposure of dioxygen to acetone solutions of 1 and 2 prepared under an anaerobic conditions at -50 degrees C. The resonance Raman spectra of the dioxygen adduct of 1 exhibited two peaks assignable to the nu(O-O) stretching mode at 873 and 887 cm(-1), which shifted to 825 and 839 cm(-1) upon binding of (18)O2. ESR spectra of all dioxygen adducts were silent. These findings suggest that dioxygen coordinates to the diiron atoms as a peroxo anion in a mu-1,2 mode. Complex 3 exhibited irreversible dioxygen binding. These results indicate that the reversible binding of dioxygen is governed by the hydrophobicity of the dioxygen-binding environment rather than the iron redox potentials.     

Kinetics of oxygen binding to ferrous myeloperoxidase

Myeloperoxidase (MPO), which is involved in host defence and inflammation, is a unique peroxidase in having a globin-like standard reduction potential of the ferric/ferrous couple. Intravacuolar and exogenous MPO released from stimulated neutrophils has been shown to exist in the oxyferrous form, called compound III. To investigate the reactivity of ferrous MPO with molecular oxygen, a stopped-flow kinetic analysis was performed. In the absence of dioxygen, ferrous MPO decays to ferric MPO (0.04 s(-1) at pH 8 versus 1.4 s(-1) at pH 5). At pH 7.0 and 25 degrees C, compound III formation (i.e., binding of dioxygen to ferrous MPO) occurs with a rate constant of (1.1+/-0.1) x 10(4)M(-1)s(-1). The rate doubles at pH 5.0 and oxygen binding is reversible. At pH 7.0, the dissociation equilibrium constant of the oxyferrous form is (173+/-12)microM. The rate constant of dioxygen dissociation from compound III is much higher than conversion of compound III to ferric MPO (which is not affected by the oxygen concentration). This allows an efficient transition of compound III to redox intermediates which actually participate in the peroxidase or halogenation cycle of MPO.     

Reaction of ferrous cytochrome c peroxidase with dioxygen: site-directed mutagenesis provides evidence for rapid reduction of dioxygen by intramolecular electron transfer from the compound I radical site.

The reaction of dioxygen with the ferrous forms of the cloned cytochrome c peroxidase [CCP(MI)] and mutants of CCP(MI) prepared by site-directed mutagenesis was studied by photolysis of the respective ferrous-CO complexes in the presence of dioxygen. Reaction of ferrous CCP(MI) with dioxygen transiently formed a FeII-O2 complex (bimolecular rate constant = (3.8 +/- 0.3) x 10(4) M-1 s-1 at pH 6.0; 23 degrees C) that reacted further (first-order rate constant = 4 +/- 1 s-1) to form a product with an absorption spectrum and an EPR radical signal at g = 2.00 that were identical to those of compound I formed by the reaction of CCP(MI)III with peroxide. Thus, the product of the reaction of CCP(MI)II with dioxygen retained three of the four oxidizing equivalents of dioxygen. Gel electrophoresis of the CCP(MI)II + dioxygen reaction products showed that covalent dimeric and trimeric forms of CCP(MI) were produced by the reaction of CCP(MI)II with dioxygen. Photolysis of the CCP(MI)II-CO complex in the presence of ferrous cytochrome c prevented the appearance of the cross-linked forms and resulted in the oxidation of 3 mol of cytochrome c/mol of CCP(MI)II-CO added. The results provide evidence that reaction of CCP(MI)II with dioxygen causes transient oxidation of the enzyme by 1 equiv above the normal compound I oxidation state. Mutations that eliminate the broad EPR signal at g = 2.00 characteristic of the compound I radical also prevented the rapid oxidation of the ferrous enzyme by dioxygen.(ABSTRACT TRUNCATED AT 250 WORDS)     

The histidine cycle: A new model for proton translocation in the respiratory heme-copper oxidases

A model of redox-linked proton translocation is presented for the terminal heme-copper oxidases. The new model, which is distinct both in principle and in detail from previously suggested mechanisms, is introduced in a historical perspective and outlined first as a set of general principles, and then as a more detailed chemical mechanism, adapted to what is known about the chemistry of dioxygen reduction in this family of enzymes. The model postulates a direct mechanistic role in proton-pumping of the oxygenous ligand on the iron in the binuclear heme-copper site through an electrostatic nonbonding interaction between this ligand and the doubly protonated imidazolium group of a conserved histidine residue nearby. In the model this histidine residue cycles between imidazolium and imidazolate states translocating two protons per event, the imidazolate state stabilized by bonding to the copper in the site. The model also suggests a key role in proton translocation for those protons that are taken up in reduction of O₂ to water, in that their uptake to the oxygenous ligand unlatches the electrostatically stabilized imidazolium residue and promotes proton release.     

Conversion of adamsite (phenarsarzin chloride) by fungal manganese peroxidase

Fungal manganese peroxidase was found to convert the persistent chemical warfare agent adamsite (phenarsarzin chloride) in a cell-free reaction mixture containing sodium malonate, Mn²⁺ ions, and reduced glutathione. The organo-arsenical compound disappeared completely within 48 h accompanied by the formation of a more polar metabolite with a clearly modified UV spectrum. Thus, As(III) in the adamsite molecule was oxidized by manganese peroxidase to As(V) which added dioxygen and released chloride.     

Determination of equilibrium constants for a sequential model of dioxygen binding by hemoglobin-inositol hexaphosphate complexes: the structural pathway from deoxy- to oxy-hemoglobin

The affinity of human hemoglobin (Hb4) for dioxygen was determined in 0.050 M bistris, 0.005 M inositol hexaphosphate (IHP) at pH 7.0 and 20.0 degrees C. Binding of dioxygen by Hb4 was determined by detailed spectroscopic analysis of the absorption spectrum in the region from 460 to 620 nm. The absorption spectrum of samples at intermediate values of fractional saturation (F) could not be resolved into components of Hb4 and (HbO2)4 without generating a residual spectrum, the amplitude of which was greatest at F from 0.4 to 0.5 and least at values of F of 0 and 1. An equation of state for dioxygen binding by the Hb4-IHP complex was formulated and tested by its ability to predict (i) the equilibrium binding curve and (ii) the variation in amplitude of the residual spectrum with F. The equilibrium binding data was fitted to the following equation of state: (Formula: see text) where K1 is the equilibrium constant for binding of dioxygen to an alpha chain of the Hb4-IHP complex, K2 is the constant for the second alpha chain, K3 is the equilibrium constant for the large-scale conformational change, K4 is the equilibrium constant for binding of oxygen by both beta chains, and (L) is the ligand concentration. The best-fitting values were as follows: K1, 0.03497 mm Hg-1; K2, 0.01368 mm Hg-1; K3, 2.44; K4, 0.0008867 mm Hg-2. The residual spectra were attributed to differential loading of dioxygen by the alpha and beta chains. Equations of state for F of each chain are presented, and the amplitude of the residual spectra was shown to be accurately predicted by the differences in F of each chain when subjected to the constraint that the best-fitting values of K1-K4 be used in predicting saturation of each chain with dioxygen.     

Eukaryotic extracellular catalase-peroxidase from Magnaporthe grisea - Biophysical/chemical characterization of the first representative from a novel phytopathogenic KatG group

All phytopathogenic fungi have two catalase–peroxidase paralogues located either intracellularly (KatG1) or extracellularly (KatG2). Here, for the first time a secreted bifunctional, homodimeric catalase–peroxidase (KatG2 from the rice blast fungus Magnaporthe grisea) has been produced heterologously with almost 100% heme occupancy and comprehensively investigated by using a broad set of methods including UV–Vis, ECD and resonance Raman spectroscopy (RR), thin-layer spectroelectrochemistry, mass spectrometry, steady-state &; presteady-state spectroscopy. RR spectroscopy reveals that MagKatG2 shows a unique mixed-spin state, non-planar heme b, and a proximal histidine with pronounced imidazolate character. At pH 7.0 and 25 °C, the standard reduction potential E°′ of the Fe(III)/Fe(II) couple for the high-spin native protein was found to fall in the range typical for the KatG family. Binding of cyanide was relatively slow at pH 7.0 and 25 °C and with a K_d value significantly higher than for the intracellular counterpart. Demonstrated by mass spectrometry MagKatG2 has the typical Trp118-Tyr251-Met277 adduct that is essential for its predominantly catalase activity at the unique acidic pH optimum. In addition, MagKatG2 acts as a versatile peroxidase using both one- and two-electron donors. Based on these data, structure–function relationships of extracellular eukaryotic KatGs are discussed with respect to intracellular KatGs and possible role(s) in host–pathogen interaction.     

The synthesis, structure and SOD-like behaviors of a μ-imidazolato-dicopper(II) complex with a binucleating hexaazamacrocycle

The synthesis, crystal structure and magnetic properties of the imidazolate-bridged dinuclear copper(II) complex [LCu₂(Im)(DMSO)₂](ClO₄)₃(DMSO), where ImH is imidazole, and L is 3, 6, 9, 17, 20, 23-hexaazatricyclo[23.3.1.1^11,13]triaconta-1(29), 11(30), 12, 14, 25(26), 27-hexaene, have been studied. Single crystal X-ray diffraction determination reveals the distorted tetragonal pyramid geometries of the imidazolate bridged dicopper(II) center are incorporated within the binucleating macrocycle. Both copper atoms are five-coordinated by four basal nitrogen atoms (three from the macrocycle and one from the imidazolate) and one oxygen atom from the DMSO molecule. Two coordinated DMSO molecules are situated at the same side of the macrocycle. The Cu–Cu separation in the complex is 5.93 Å. Magnetic measurements and ESR spectroscopy studies exibit an antiferromagnetic exchange interaction with a coupling constant of J= − 26.94 cm⁻¹. Investigations on the pH-dependent ESR of the title compound in frozen 50% aqueous DMSO solution confirm the existence of the bridged cation [LCu₂(Im)]³⁺ as a major species in solution mainly in the range 5.2pH11.5. The imidazolate bridge is broken in the pH range of 5.2–3.3, which is close to that for the native enzyme.     

Modulation of the NO<Emphasis Type="Italic"> trans</Emphasis> effect in heme proteins: implications for the activation of soluble guanylate cyclase

The binding of NO to the iron heme in guanylate cyclase and other heme proteins induces the cleavage of the proximal histidine bonded to the metal. In this study we assess by means of density functional theory (DFT) electronic structure calculations the role of H-bonding to histidine in the modulation of this effect. We have considered in the first place a model of the isolated active site coordinated with imidazole and imidazolate to mimic the effects of a very strong H-bond. We have also investigated four selected ferrous heme proteins with different proximal histidine environments: the O(2) sensing FixL, horseradish peroxidase C, and the alpha and beta subunits of human hemoglobin. Our results indicate that polarization and charge transfer effects associated with H-bonding to the proximal histidine play a fundamental role in the modulation of the NO trans effect in heme proteins. We also find computational evidence suggesting that protein structural constraints may affect significantly the cleavage of the Fe-His bond.     

Deuterium exchangeable proton hyperfine resonances of low-spin cytochrome c peroxidase and the mechanism of peroxidase catalysis

Deuterium exchangeable hyperfine proton NMR resonances of cytochrome c peroxidase (EC 1.11.1.5) are identified in H2O solutions of the enzyme. One of these is assigned to the proximal histidine's imidazole N-H. Its shift and pH dependence indicate that an imidazolate form, which has been postulated for peroxidases, is ruled out for cytochrome c peroxidase-cyanide. A qualitative comparison of relative heme-pocket dynamics is also possible. When the bulk water resonance is irradiated with a continuous, but off acquisition, decoupler frequency the N-H resonance shows no intensity loss, indicating that saturation transfer between the proximal histidine and solvent water is either minimal, or extremely slow.     

Synthetic analogue approach to metallobleomycins: syntheses, structure and properties of mononuclear and tetranuclear gallium(III) complexes of a ligand that resembles the metal-binding site of bleomycin

As part of our interest into the bioinorganic chemistry of gallium, gallium(III) complexes of the peptide ligand N-(2-(4-imidazolyl)ethyl)pyridine-2-carboxamide (pypepH2) resembling a fragment of the metal-binding domain of bleomycins (BLMs), have been isolated. Reaction of pypepH2 with (Et4N)[GaCl4] and Ga(acac)3 [acac- is the acetylacetonate(-1) ion] affords the mononuclear complex [Ga(pypepH)2]Cl.2H2O (1) and the tetranuclear complex [Ga4(acac)4(pypep)4].4.4H2O (2), respectively. Both complexes were characterized by single-crystal X-ray crystallography, IR spectroscopy and thermal decomposition data. The pypepH- ion in 1 behaves as a N(pyridyl), N(deprotonated amide), N(pyridine-type imidazole) chelating ligand. The doubly deprotonated pypep2- ion in 2 behaves as a N(pyridyl), N(deprotonated amide), N(imidazolate), N'(imidazolate) mu2 ligand and binds to one Ga(III) atom at its pyridyl, amide and one of the imidazolate nitrogens, and to a second metal ion at the other imidazolate nitrogen; a chelating acac- ligand completes six coordination at each Ga(III) centre. The IR data are discussed in terms of the nature of bonding and known structures. The 1H NMR spectrum of 1 suggests that the cation of the complex maintains its integrity in dimethylsulfoxide (DMSO) solution. Complexes 1 and 2 are the first synthetic analogues of metallobleomycins with gallium(III).     

Disruption of an active site hydrogen bond converts human heme oxygenase-1 into a peroxidase

The crystal structure of heme oxygenase-1 suggests that Asp-140 may participate in a hydrogen bonding network involving ligands coordinated to the heme iron atom. To examine this possibility, Asp-140 was mutated to an alanine, phenylalanine, histidine, leucine, or asparagine, and the properties of the purified proteins were investigated. UV-visible and resonance Raman spectroscopy indicate that the distal water ligand is lost from the iron in all the mutants except, to some extent, the D140N mutant. In the D140H mutant, the distal water ligand is replaced by the new His-140 as the sixth iron ligand, giving a bis-histidine complex. The D140A, D140H, and D140N mutants retain a trace (<3%) of biliverdin forming activity, but the D140F and D140L mutants are inactive in this respect. However, the two latter mutants retain a low ability to form verdoheme, an intermediate in the reaction sequence. All the Asp-140 mutants exhibit a new peroxidase activity. The results indicate that disruption of the distal hydrogen bonding environment by mutation of Asp-140 destabilizes the ferrous dioxygen complex and promotes conversion of the ferrous hydroperoxy intermediate obtained by reduction of the ferrous dioxygen complex to a ferryl species at the expense of its normal reaction with the porphyrin ring.     

Preparation of dehydro-l-(+)-ascorbic acid dimer by oxidation of ascorbic acid with arsenic acid/iodine and formation of complexes between arsenious acid and ascorbic acid

Ascorbic acid in the presence of a catalytic amount of iodine reduces arsenic acid in methanol giving the arsenious acid bound to the 2-methyl hemi-ketal of dehydroascorbic acid, 5, in 1:1 and in a more stable 2:1 5/As(III) molar ratio. Removal of the As(III) and treating the 2-methyl hemi-ketal of dehydroascorbic acid with refluxing acetonitrile affords the pure, crystalline dehydroascorbic acid dimer in good yields. Ascorbic acid also binds to As(III) of H(3)AsO(3) in a 1:1 and 2:1 ascorbic acid/As(III) molar ratio. The 1:1 complex is not stable and by expulsion of H(3)AsO(3) is transformed to the more stable 2:1 complex. The data do not permit distinguishing the 2:1 complexes between [AsL(2)(H(2)O)](-)H(+) or AsL(LH)(H(2)O) where L is the bis deprotonated and LH is the mono deprotonated 2-methyl hemi-ketal of dehydroascorbic acid or ascorbic acid. The 2:1 ascorbic acid/As(III) complex is oxidized by dioxygen, in a solvent-dependent manner, to dehydroascorbic acid implying dioxygen activation by the bound As(III). With thiophenol the same complex gives quantitatively triphenyl trithioarsenite, As(SPh)(3).     

Crystal structures of the ferrous dioxygen complex of wild-type cytochrome P450eryF and its mutants, A245S and A245T: investigation of the proton transfer system in P450eryF

Cytochrome P450eryF (CYP107A) from Saccaropolyspora ertherea catalyzes the hydroxylation of 6-deoxyerythronolide B, one of the early steps in the biosynthesis of erythromycin. P450eryF has an alanine rather than the conserved threonine that participates in the activation of dioxygen (O(2)) in most other P450s. The initial structure of P450eryF (Cupp-Vickery, J. R., Han, O., Hutchinson, C. R., and Poulos, T. L. (1996) Nat. Struct. Biol. 3, 632-637) suggests that the substrate 5-OH replaces the missing threonine OH group and holds a key active site water molecule in position to donate protons to the iron-linked dioxygen, a critical step for the monooxygenase reaction. To probe the proton delivery system in P450eryF, we have solved crystal structures of ferrous wild-type and mutant (Fe(2+)) dioxygen-bound complexes. The catalytic water molecule that was postulated to provide protons to dioxygen is absent, although the substrate 5-OH group donates a hydrogen bond to the iron-linked dioxygen. The hydrogen bond network observed in the wild-type ferrous dioxygen complex, water 63-Glu(360)-Ser(246)-water 53-Ala(241) carbonyl in the I-helix cleft, is proposed as the proton transfer pathway. Consistent with this view, the hydrogen bond network in the O(2).A245S and O(2) .A245T mutants, which have decreased or no enzyme activity, was perturbed or disrupted, respectively. The mutant Thr(245) side chain also perturbs the hydrogen bond between the substrate 5-OH and dioxygen ligand. Contrary to the previously proposed mechanism, these results support the direct involvement of the substrate in O(2) activation but raise questions on the role water plays as a direct proton donor to the iron-linked dioxygen.     

Oxygen, oxidases, and the essential trace metals

The dominant function of dioxygen as the terminal electron acceptor in aerobic systems is well established; the roles of iron and copper in the terminal oxidases are less well understood. The minor, but crucial, part that dioxygen plays in other biological processes has recently attracted much attention. The chemistry of the reduction products of dioxygen is described and the possible relation of these products to the toxic properties of dioxygen is discussed. It is suggested that the uncontrolled reaction of dioxygen with reduced species, to give the superoxide ion, hydrogen peroxide, the hydroxyl radical and perhaps other entities derived from these, is potentially hazardous to the organism. Defences exist against these species, not least in the dismutases dependent on copper-zinc, manganese and iron, in catalase and in the selenium-dependent peroxidase. The effectiveness of these defences is examined and their reduction products of dioxygen during phagocytosis is discussed.     

Binding of dioxygen to non-metal sites in proteins: exploration of the importance of binding site size versus hydrophobicity in the copper amine oxidase from Hansenula polymorpha

Copper amine oxidases (CAOs) contain 2,4,5-trihydroxyphenylalanyl quinone (TPQ) and a copper ion in their active sites, catalyzing amine oxidation to aldehyde and ammonia concomitant with the reduction of molecular oxygen to hydrogen peroxide. Kinetic studies on the CAO from bovine serum (BSAO) [Su and Klinman (1999) Biochemistry 37, 12513-12525] and the recent reports on the cobalt substituted form of the enzyme from Hansenula polymorpha (HPAO) [Mills and Klinman (2000) J. Am. Chem. Soc. 122, 9897-9904, and Mills et al. (2002) Biochemistry, 41, 10577-10584] support pre-binding of molecular oxygen prior to a rate-limiting electron transfer from the reduced form of TPQ (p-aminohydroquinone form) to dioxygen. Although there is significant sequence homology between BSAO and HPAO, k(cat)/K(m)(O2) for BSAO under the optimal condition is one order of magnitude lower than that for HPAO. From a comparison of amino acid sequences for BSAO and HPAO, together with the X-ray crystal structure of HPAO, a plausible dioxygen pre-binding site has been identified that involves Y407, L425, and M634 in HPAO; the latter two residues are altered in BSAO to A490 and T695. To determine which of these residues plays a greater role in dioxygen chemistry, k(cat)/K(m)(O2) was determined in HPAO for the M634 --> T and L425 --> A mutants. The L425 --> A mutation does not alter k(cat)/K(m)(O2) to a large extent, whereas the M634 --> T decreased k(cat)/K(m)(O2) by one order of a magnitude, creating a catalyst that is similar to BSAO. A series of mutants at M634 (to F, L, and Q) were, therefore, prepared in HPAO and characterized with regard to k(cat)/K(m)(O2) as a function of pH. Structure reactivity correlations show a linear relationship of rate with side chain volume, rather than hydrophobicity, indicating that dioxygen reactivity increases with the bulk of the residue at position 634. This site also shows specificity for O2, in relation to the co-gas N2, since substitution of the inert gas N2 by either Ar or He has no effect on measured rates. In particular, He gas is expected to have little affinity for protein at 1 atmospheric pressure, implying little or no binding by N2 as well.     

Role of active site water molecules and substrate hydroxyl groups in oxygen activation by cytochrome P450 158A2: a new mechanism of proton transfer

From the x-ray crystal structure of CYP158A2 (Zhao, B., Guengerich, F. P., Bellamine, A., Lamb, D. C., Izumikawa, M., Lei, L., Podust, L. M., Sundaramoorthy, M., Reddy, L. M., Kelly, S. L., Kalaitzis, J. A., Stec, D., Voehler, M., Falck, J. R., Moore, B. S., Shimada, T., and Waterman, M. R. (2005) J. Biol. Chem. 280, 11599-11607), one of 18 cytochrome P450 (CYP) genes in the actinomycete Streptomyces coelicolor, ordered active site water molecules (WAT505, WAT600, and WAT640), and hydroxyl groups of the substrate flaviolin were proposed to participate in proton transfer and oxygen cleavage in this monooxygenase. To probe their roles in catalysis, we have studied the crystal structures of a substrate analogue (2-hydroxy-1,4-naphthoquinone) complex with ferric CYP158A2 (2.15 A) and the flaviolin ferrous dioxygen-bound CYP158A2 complex (1.8 A). Catalytic activity toward 2-hydroxy-1,4-naphthoquinone was approximately 70-fold less than with flaviolin. In the ferrous dioxygen-bound flaviolin complex, the three water molecules in the ferric flaviolin complex still occupy the same positions and form hydrogen bonds to the distal dioxygen atom. These findings suggest that CYP158A2 utilizes substrate hydroxyl groups to stabilize active site water and further assist in the iron-linked dioxygen activation. A continuous hydrogen-bonded water network connecting the active site to the protein surface (bulk solvent) not present in the other two ferrous dioxygen-bound P450 structures (CYP101A1/P450cam and CYP107A1/P450eryF) is proposed to participate in the proton-delivery cascade, leading to dioxygen bond scission. This ferrous-dioxygen structure suggests two classes of P450s based on the pathway of proton transfer, one using the highly conserved threonine in the I-helix (CYP101A1) and the other requiring hydroxyl groups of the substrate molecules either directly transferring protons (CYP107A1) or stabilizing a water pathway for proton transfer (CYP158A2).