Roman Davydov

EPR and ENDOR Characterization of the Reactive Intermediates in the Generation of NO by Cryoreduced Oxy-Nitric Oxide Synthase from Geobacillus stearothermophilus§

Cryoreduction EPR/ENDOR/step-annealing measurements with substrate complexes of oxy-gsNOS (3; gsNOS is nitric oxide synthase from Geobacillus stearothermophilus) confirm that Compound I (6) is the reactive heme species that carries out the gsNOS-catalyzed (Stage I) oxidation of L-arginine to N-hydroxy-L-arginine (NOHA), whereas the active species in the (Stage II) oxidation of NOHA to citrulline and HNO/NO(-) is the hydroperoxy-ferric form (5). When 3 is reduced by tetrahydrobiopterin (BH4), instead of an externally supplied electron, the resulting BH4(+) radical oxidizes HNO/NO(-) to NO. In this report, radiolytic one-electron reduction of 3 and its complexes with Arg, Me-Arg, and NO(2)Arg was shown by EPR and (1)H and (14,15)N ENDOR spectroscopies to generate 5; in contrast, during cryoreduction of 3/NOHA, the peroxo-ferric-gsNOS intermediate (4/NOHA) was trapped. During annealing at 145 K, ENDOR shows that 5/Arg and 5/Me-Arg (but not 5/NO(2)Arg) generate a Stage I primary product species in which the OH group of the hydroxylated substrate is coordinated to Fe(III), characteristic of 6 as the active heme center. Analysis shows that hydroxylation of Arg and Me-Arg is quantitative. Annealing of 4/NOHA at 160 K converts it first to 5/NOHA and then to the Stage II primary enzymatic product. The latter contains Fe(III) coordinated by water, characteristic of 5 as the active heme center. It further contains quantitative amounts of citrulline and HNO/NO(-); the latter reacts with the ferriheme to form the NO-ferroheme upon further annealing. Stage I delivery of the first proton of catalysis to the (unobserved) 4 formed by cryoreduction of 3 involves a bound water that may convey a proton from L-Arg, while the second proton likely derives from the carboxyl side chain of Glu 248 or the heme carboxylates; the process also involves proton delivery by water(s). In the Stage II oxidation of NOHA, the proton that converts 4/NOHA to 5/NOHA likely is derived from NOHA itself, a conclusion supported by the pH invariance of the process. The present results illustrate how the substrate itself modulates the nature and reactivity of intermediates along the monooxygenase reaction pathway.

Mechanistic enzymology of oxygen activation by the cytochromes P450.

The P450 cytochromes represent a universal class of heme-monooxygenases. The detailed mechanistic understanding of their oxidative prowess is a critical theme in the studies of metabolism of a wide range of organic compounds including xenobiotics. Integral to the O2 bond cleavage mechanism by P450 is the enzymes concerted use of protein and solvent-mediated proton transfer events to transform reduced dioxygen to a species capable of oxidative chemistry. To this end, a wide range of kinetic, structural, and mutagenesis data has been accrued. A critical role of conserved acid-alcohol residues in the P450 distal pocket, as well as stabilized waters, enables the enzyme to catalyze effective monooxygenation chemistry. In this review, we discuss the detailed mechanism of P450 dioxygen scission utilizing the CYP101 hydroxylation of camphor as a model system. The application of low-temperature radiolytic techniques has enabled a structural and spectroscopic analysis of the nature of critical intermediate states in the reaction.

Characterization of the microsomal cytochrome P450 2B4 O2 activation intermediates by cryoreduction and electron paramagnetic resonance

The oxy-ferrous complex of cytochrome P450 2B4 (2B4) has been prepared at -40 degrees C with and without bound substrate [butylated hydroxytoluene (BHT)] and radiolytically one-electron cryoreduced at 77 K. Electron paramagnetic resonance (EPR) shows that in both cases the observed product of cryoreduction is the hydroperoxo-ferriheme species, indicating that the microsomal P450 contains an efficient distal-pocket proton-delivery network. In the absence of substrate, two distinct hydroperoxo-ferriheme signals are observed, reflecting the presence of two major conformational substates in the oxy-ferrous precursor. Only one species is observed when BHT is bound, indicating a more ordered active site. BHT binding also changes the g-tensor components of the hydroperoxo-ferric 2B4 intermediate, indicating that the substrate modulates the properties of this intermediate. Step annealing the cryoreduced ternary 2B4 complex at >or=175 K causes the loss of hydroperoxo-ferric 2B4 and the parallel appearance of high-spin ferric 2B4; liquid chromatography-tandem mass spectroscopy (LC-MS/MS) analysis shows that in this process BHT is quantitatively converted to two products, hydroxymethyl BHT (1) and 3-hydroxy- tert-butyl BHT (2). This implies that the hydroperoxo-ferric 2B4 prepared by cryoreduction is catalytically active and that the high-spin state observed after annealing contains an enzyme-bound product of BHT monooxygenation. The ratio of products generated during cryoreduction and annealing (6.2/1) is significantly different from the ratio (2.5/1) at ambient temperature. These findings suggest that substrate is held more rigidly relative to the oxidizing species at low temperatures and/or that dissociation of FeOOH is inhibited at low temperature. As in experiments under ambient conditions, product formation is not observed with the inactive F429H 2B4 mutant.

Active intermediates in heme monooxygenase reactions as revealed by cryoreduction/annealing, EPR/ENDOR studies

This review describes the use of cryoreduction/annealing EPR/ENDOR techniques for determining the active oxidizing species in reactions catalyzed by heme monooxygenases. The three candidate heme states are: ferric peroxo, ferric hydroperoxo and compound I intermediates. The enzymes discussed include cytochromes P450, nitric oxide synthase and heme oxygenase.

Probing the ternary complexes of indoleamine and tryptophan 2,3-dioxygenases by cryoreduction EPR and ENDOR spectroscopy

We have applied cryoreduction/EPR/ENDOR techniques to characterize the active-site structure of the ferrous-oxy complexes of human (hIDO) and Shewanella oneidensis (sIDO) indoleamine 2,3-dioxygenases, Xanthomonas campestris (XcTDO) tryptophan 2,3-dioxygenase, and the H55S variant of XcTDO in the absence and in the presence of the substrate l-Trp and a substrate analogue, l-Me-Trp. The results reveal the presence of multiple conformations of the binary ferrous-oxy species of the IDOs. In more populated conformers, most likely a water molecule is within hydrogen-bonding distance of the bound ligand, which favors protonation of a cryogenerated ferric peroxy species at 77 K. In contrast to the binary complexes, cryoreduction of all of the studied ternary [enzyme-O2-Trp] dioxygenase complexes generates a ferric peroxy heme species with very similar EPR and 1H ENDOR spectra in which protonation of the basic peroxy ligand does not occur at 77 K. Parallel studies with l-Me-Trp, in which the proton of the indole nitrogen is replaced with a methyl group, eliminate the possibility that the indole NH group of the substrate acts as a hydrogen bond donor to the bound O2, and we suggest instead that the ammonium group of the substrate hydrogen-bonds to the dioxygen ligand. The present data show that substrate binding, primarily through this H-bond, causes the bound dioxygen to adopt a new conformation, which presumably is oriented for insertion of O2 into the C2−C3 double bond of the substrate. This substrate interaction further helps control the reactivity of the heme-bound dioxygen by “shielding” it from water.

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.

Studies on an iron(III)-peroxo porphyrin. Iron(III)-peroxo or iron(II)-superoxo?

We demonstrate that a one electron reduced product of the heme iron dioxygen adduct exists in solution not only as the commonly accepted iron(iii)-peroxo species, but coexists with its isomeric iron(ii)-superoxo form. This unusual reduced metal-superoxide adduct [M(ii)-O(2)(-)] is recently reported as a reactive intermediate in the case of non-heme extradiol dioxygenases and could also be generated by cryoreduction of a heme Fe(II)-O(2) adduct. The existence of iron(ii)-superoxo species in solution is consistent with IR, EPR, mass and Mössbauer spectra. The equilibrium between heme iron(iii)-peroxo and iron(ii)-superoxo forms is supported by density functional theory and explains our previous finding that upon release of coordinated (su)peroxide a corresponding iron(ii) complex remains. These results shed new light on the nature of heme iron(iii)-peroxo species that are key intermediates in the metalloenzyme-catalyzed dioxygen and hydrogen peroxide activation.

Characterization and structure of a Zn2+ and [2Fe-2S]-containing copper chaperone from Archaeoglobus fulgidus.

Bacterial CopZ proteins deliver copper to P1B-type Cu+-ATPases that are homologous to the human Wilson and Menkes disease proteins. The genome of the hyperthermophile Archaeoglobus fulgidus encodes a putative CopZ copper chaperone that contains an unusual cysteine-rich N-terminal domain of 130 amino acids in addition to a C-terminal copper binding domain with a conserved CXXC motif. The N-terminal domain (CopZ-NT) is homologous to proteins found only in extremophiles and is the only such protein that is fused to a copper chaperone. Surprisingly, optical, electron paramagnetic resonance, and x-ray absorption spectroscopic data indicate the presence of a [2Fe-2S] cluster in CopZ-NT. The intact CopZ protein binds two copper ions, one in each domain. The 1.8Å resolution crystal structure of CopZ-NT reveals that the [2Fe-2S] cluster is housed within a novel fold and that the protein also binds a zinc ion at a four-cysteine site. CopZ can deliver Cu+ to the A. fulgidus CopA N-terminal metal binding domain and is capable of reducing Cu2+ to Cu+. This unique fusion of a redox-active domain with a CXXC-containing copper chaperone domain is relevant to the evolution of copper homeostatic mechanisms and suggests new models for copper trafficking.

The Use of Deuterated Camphor as a Substrate in 1H ENDOR Studies of Hydroxylation by Cryoreduced Oxy P450cam Provides New Evidence of the Involvement of Compound I

Electron paramagnetic resonance and (1)H electron nuclear double resonance (ENDOR) spectroscopies have been used to analyze intermediate states formed during the hydroxylation of (1R)-camphor (H(2)-camphor) and (1R)-5,5-dideuterocamphor (D(2)-camphor) as induced by cryoreduction (77 K) and annealing of the ternary ferrous cytochrome P450cam-O(2)-substrate complex. Hydroxylation of H(2)-camphor produced a primary product state in which 5-exo-hydroxycamphor is coordinated with Fe(III). ENDOR spectra contained signals derived from two protons [Fe(III)-bound C5-OH(exo) and C5-H(endo)] from camphor. When D(2)-camphor was hydroxylated under the same condition in H(2)O or D(2)O buffer, both ENDOR H(exo) and H(endo) signals are absent. For D(2)-camphor in H(2)O buffer, H/D exchange causes the C5-OH(exo) signal to reappear during relaxation upon annealing to 230 K; for H(2)-camphor in D(2)O, the magnitude of the C5-OH(exo) signal decreases via H/D exchange. These observations clearly show that Compound I is the reactive species in the hydroxylation of camphor in P450cam.

Radiolytic Reduction of Methane Monooxygenase Dinuclear Iron Cluster at 77 K EPR EVIDENCE FOR CONFORMATIONAL CHANGE UPON REDUCTION OR BINDING OF COMPONENT B TO THE DIFERRIC STATE

The soluble form of methane monooxygenase (MMO) consists of three components: reductase, hydroxylase (MMOH), and “B” (MMOB). Resting MMOH contains a diferric bis-μ-hydroxodinuclear iron “diamond core” cluster which is the site of oxygen activation chemistry after reduction. Here it is shown that γ-irradiation of MMOH at 77 K results in reduction of the diiron cluster to an EPR active Fe(II)·Fe(III) mixed valence state. At this temperature, the conformation of the enzyme remains essentially unchanged during reduction, so the EPR-spectrum becomes a probe of the conformation of the diferric state. The γ-irradiated MMOH exhibits EPR spectra that differ in lineshape and saturation properties from those of the mixed valence MMOH generated by chemical reduction in solution; annealing the γ-irradiated sample at 230 K yields the spectra of the chemically reduced sample. This demonstrates that the conformation of diferric MMOH in the vicinity of the diiron cluster changes during reduction to the mixed valence state. The analogous experiment for the MMOB·MMOH complex gives a new mixed valence species following γ-irradiation that differs from all previously reported mixed valence species. Thus, binding of MMOB also causes a change in the conformation of diferric MMOH. It is hypothesized that the structural changes observed for the first time here may involve conversion of the dihydroxo-bridged diamond core structure to one with more readily dissociable bridging oxygen ligands to facilitate reaction with O2 following cluster reduction.