Environmental effects on primary radical formation in guanine: solid-state ESR and ENDOR of guanine hydrobromide monohydrate.

Single crystals of guanine hydrobromide monohydrate, in which the guanine base is protonated at N7, were X-irradiated at 8 and 65 K. Using K-band ESR, ENDOR, and field-swept-ENDOR (FSE) techniques, the crystals were studied between 8 K and room temperature. There was evidence for five different radicals, RI-RV, immediately following irradiation at 8 or 65 K. RI was identified as the O6-protonated anion. It decayed near room temperature with no detectable successor. RII was identified as the N7-deprotonated cation, and decayed near 130 K. RIII is thought to be a ring-opened product formed by C8-N9 bond rupture; upon warming, it decayed at 150 K. RIV is the well-known C8 H-addition radical. These four radicals have been observed previously in the hydrochloride salt of guanine monohydrate. RV is novel, however, with magnetic characteristics consistent with those of the product formed by net OH addition to C5 of the unsaturated C4-C5 bond. It is characterized by four alpha-proton couplings indicating pi-electron spin as follows: 13% at C8; 11% at N7; and 12% at N10. RV decayed between 240 and 255 K with no detectable successor. Upon further warming, very weak resonance lines due to additional, unidentified radicals were observed. A comparison of these results with those obtained from other systems containing N7-protonated guanine bases demonstrates the effect of the environment on the primary radical formation.     

Free radical formation in single crystals of 2'-deoxyguanosine 5'-monophosphate tetrahydrate disodium salt: an EPR/ENDOR study.

Single crystals of 2'-deoxyguanosine 5'-monophosphate were X-irradiated at 10 K and at 65 K, receiving doses between 4.5 and 200 kGy, and studied using K-band EPR, ENDOR, and field-swept ENDOR (FSE) spectroscopy. Evidence for five base-centered and more than nine sugar-centered radicals was found at 10 K following high radiation doses. The base-centered radicals were the charged anion, the N10-deprotonated cation, the C8 H-addition radical, a C5 H-addition radical, and finally a stable radical so far unidentified but with parameters similar to those expected for the charged cation. The sugar-centered radicals were the H-abstraction radicals centered at C1', C2', C3', and C5', an alkoxy radical centered at O3', a C5'-centered radical in which the C5'-O5' phosphoester bond appears to be ruptured, a radical tentatively assigned to a C4'-centered radical involving a sugar-ring opening, as well as several additional unidentified sugar radicals. Most radicals were formed regardless of radiation doses. All radicals formed following low doses (4.5-9 kGy) were also observed subsequent to high doses (100-200 kGy). The relative amount of some of the radicals was dose dependent, with base radicals dominating at low doses, and a larger relative yield of sugar radicals at high doses. Above 200 K a transformation from a sugar radical into a base radical occurred. Few other radical transformations were observed. In the discussion of primary radicals fromed in DNA, the presence of sugar-centered radicals has been dismissed since they are not apparent in the EPR spectra. The present data illustrate how radicals barely traceable in the EPR spectra may be identified due to strong ENDOR resonances. Also, the observation of a stable radical with parameters similar to those expected for the charge guanine cation is interesting with regard to the nature of the primary radicals stabilized in X-irradiated DNA.     

Radiation-induced hydroxyl addition to purine molecules: EPR and ENDOR study of hypoxanthine hydrochloride monohydrate single crystals

Three radical species were detected in an EPR/ENDOR study of X-irradiated hypoxanthine.HCl.H2O single crystals at room temperature: RI was identified as the product of net H addition to C8, RII was identified as the product of net H addition to C2, and RIII was identified as the product of OH addition to C8. The observed set of radicals was the same for room-temperature irradiation as for irradiation at 10 K followed by warming the crystals to room temperature; however, the C2 H-addition and C8 OH-addition radicals were not detectable after storage of the crystals for about 2 months at room temperature. Use of selectively deuterated crystals permitted unique assignment of the observed hyperfine couplings, and results of density functional theory calculations on each of the radical structures were consistent with the experimental results. Comparison of these experimental results with others from previous crystal-based systems and model system computations provides insight into the mechanisms by which the biologically important purine C8 hydroxyl addition products are formed. The evidence from solid systems supports the mechanism of net water addition to one-electron oxidized purine bases and demonstrates the importance of a facial approach between the reactants.     

Affinity modification of membrane-bound microsomal cytochrome P-450 with lipophilic analogs of substrates

The following lipophilic spin-labeled cytochrome P-450 analogs were synthesized: 2-octyl-4-(3-iodine-2-oxopropylidene)-2,3,5,5-tetramethylimidaz olidine-1-oxyl (RIII), 2-nonyl-4-(3-iodine-2-oxopropylidene)-2,3,5,5-tetramethylimidaz olidine-1-oxyl (RIV), 2-hepta-decyl-4-(3-iodine-2-oxopropylidene)-2,3,5,5-tetramethyl imidazolidine-1- oxyl (RV). The distribution coefficients, k, in water--lipid and water--octanol systems as well as the theoretical estimates of k for these and previously synthesized analogs, i.e., 4-(3-iodine-2-oxo-propylidenyl)-2,2,3,5,5-pentamethylimidazolid ine-1-oxyl (RI) and 2-hexyl-4-(3-iodine-2-oxopropylidene)-2,3,5,5-tetramethylimidaz olidine- 1-oxyl (RII) were determined. It was shown that RIII and RIV bind as type I substrates to cytochrome P-450 from rat microsomes induced with phenobarbital or 3-methylcholanthrene as well as to those from control rats. Radicals RIII and RIV inhibit the oxidation of aniline, aminopyrine and benzphetamine. RIII-RV strongly inhibit the O-deethylation of 7-etoxyresorufin. The inhibitory activity of the radicals increases in the following order: RV less than RIV less than or equal to RI less than or equal to RIII less than RII. The experimental results suggest that the inhibitory properties are nonmonotonuesly related to the lipophility. The high lipophility of RIII and its strong inhibitory properties permit to render the latter to the class of inhibitors which can be transported by liposome membrane vehicles to the liver, inhibit the in vivo activity of the microsomal system and thus prolong the effects of drugs oxidized by cytochrome P-450.     

Free radical formation in X-irradiated crystals of 2'-deoxycytidine hydrochloride. Electron magnetic resonance studies at 10 K

Single crystals of deoxycytidine hydrochloride (CdR.HCl) have been X-irradiated at 10 K with doses up to about 150 kGy and studied using 24 GHz (K-band) EPR, ENDOR and FSE spectroscopy. In this system, the cytosine base is protonated at the N3 position. Nine different radicals were characterized and identified. Three of these are ascribed to three versions of the one-electron reduced species, probably differing in their protonation state. Radicals formed by net hydrogen addition to the cytosine C5 and C6 positions were observed at 10 K. The hydrogen-abstraction radical at the deoxyribose C1' position most probably results from initial oxidation of the base. The remaining radical species are all localized to the sugar moiety, representing products formed by net hydrogen abstraction from three of the five available carbons of the deoxyribose sugar. The lack of base-centered oxidation products as well as the structures of the one-electron reduced species is rationalized by considering the specific proton donor-acceptor properties of this crystalline lattice in comparison with similar systems.     

ESR/ENDOR study of guanosine 5'-monophosphate (free acid) single crystals X-irradiated at 10 K

Single crystals of the free base of guanosine 5'-monophosphate were X-irradiated at 10 and at 65 K and investigated between these temperatures and room temperature using K-band ESR and ENDOR spectroscopy. Three free radicals were detected in this temperature range. Two of these were identified as the O6-protonated anion radical and the C8 H-addition radical. Both of these species were present immediately after irradiation at 10 K. The anion radical was formed in two slightly different conformations, of which one decayed at about 150 K and the second at about 250 K. No successor radicals could be detected following the decay of the anion radical. The C8 H-adduct was stable at all temperatures used. The use of partially deuterated crystals confirmed the assignments made and showed that the main pathway for the formation of the C8 H-adduct consisted of addition of a proton from an easily exchangeable site. It is suggested that the C8 H-adduct is formed subsequent to a primary oxidation event localized either at the guanine base or at a nearby water of crystallization. Possible mechanisms for the formation of this product are discussed.     

EPR and ENDOR study of crystalline cytosine x HCl doped with 5-methylcytosine. Radiation-induced radical formation and hole transfer

Radical formation and hole transfer were investigated in crystals of cytosine.HCl (C.HCl) doped with 0-1.1 mol-% 5-methylcytosine x HCl (5MC x HCl). The doping level was determined by NMR spectroscopy. Crystals and polycrystalline samples were X-irradiated at 295 K, 77 K and 12 K and studied with EPR, ENDOR and FSE spectroscopy at these temperatures. At 295 K the dominant radicals were the so-called 3alphaH radical, formed in 5MC by a net H-abstraction from the methyl group, and the cytosine C6 H-addition (5-yl) radical. At 12 K five radicals were identified. These were the 3alphaH radical, cytosine reduction and oxidation products, and the cytosine C6 and C5 H-addition (5-yl and 6-yl, respectively) radicals. The spectroscopic parameters for the 3alphaH radical are very similar to those of a radical observed previously in the crystalline cytosine derivatives cytidine (CR), 2'deoxycytidine hydrochloride (CdR x HCl), 5'dCMP and 3'CMP as well as in the uracil derivative 2-thiouracil (2-TU). It was shown that amounts of the order of tenths of a percent 5MC x HCl doped into crystals of C.HCl give rise to a considerable yield of 3alphaH radicals after exposure to ionizing radiation both at room temperature and at lower temperatures. This supports a previous suggestion that naturally occurring 5-methylated cytosine impurities may be responsible for the formation of 3alphaH radicals in the crystalline cytosine derivatives CR, CdR.HCl, 5'dCMP and 3'CMP and suggests that the 3alphaH radical in these systems is a 5-methylated base-centered radical. The total radical yield in doped C x HCl crystals increased considerably with the doping level, both at low temperatures and at room temperature, implying that the 3alphaH radical is more stable than the primary cytosine radicals. The relative amounts of the 3alphaH radical were obtained by using simulated benchmark spectra to reconstruct experimental EPR spectra of doped polycrystalline samples. Evidence is presented suggesting that the enhanced yield of the 3alphaH radical in doped samples is due to holes originally formed at cytosine bases and transferred to 5-methylcytosine bases in addition to the 3alphaH radical being less exposed to recombination than other cytosine radicals.     

ESR and ENDOR study of adenosine single crystals X-irradiated at 10 K

Single crystals of adenosine were X-irradiated at 10 K and investigated between 10 and 300 K using K-band ESR and ENDOR spectroscopy. Two free radicals were analyzed. Radical I exhibits small hyperfine couplings to the C8-H, C2-H, and a N3-H protons, and was identified as the N3 protonated base anion radical. Radical II exhibits small hyperfine couplings to a C8-H and an exocyclic -N10-H proton. It is suggested that this is therefore the N10 deprotonated base cation radical. Enough data were not available to analyze a third primary radical believed to be located on the ribose moiety. Upon warming Radical I decays at ca. 40 K with no apparent successor. Likewise, no successor was identified for Radical II, which decays at ca. 100 K. At ca. 200 K there is ESR evidence for the C2 and C8 H-addition radicals. Their precursors have not been identified.     

Free radical formation in crystals of 2'-deoxyguanosine 5'-monophosphate irradiated at 15 K: an ESR study

Radiation-induced radicals in single crystals of 2'-deoxyguanosine 5'-monophosphate (5'-dGMP) at 15 K have been studied by electron spin resonance (ESR) spectroscopy. At low temperatures three radicals were analyzed in detail. The negatively charged pi anion of the guanine base completely dominated the spectra. Weaker resonances were due to an alkoxy radical with the spin density in the C3'-O3' region of the sugar moiety as well as another sugar-centered radical. The anion rapidly decayed upon exposure to uv light at 15 K or by annealing above 25 K. In both cases no successor radical was observed. The second sugar-centered radical decays at 200 K with a concomitant appearance of the resonance from the C8 H-addition radical. By annealing at 295 K the latter resonance was the only one observed. After irradiation at 295 K, however, an additional resonance from a sugar-centered radical, which has been analyzed previously by B. Rakvin and J. N. Herak (Radiat. Res. 88, 240-250 (1981)) was observed. A reinvestigation of this resonance was performed.     

Electron trapping and reactions in rhamnose by ESR and ENDOR.

The main objective for a reinvestigation of rhamnose was to devise a mechanistic link between the trapped electron detected previously and the secondary radicals observed at 77 K and at room temperature. Single crystals of rhamnose were X-irradiated at temperatures between 15 and 300 K and examined using ESR, ENDOR, and field-swept ENDOR techniques. After low-temperature irradiation a C3 H-abstraction radical is formed following the visible light-induced decay of the trapped electron. This species was previously assigned erroneously to a C2 H-abstraction species. At temperatures above 120 K, this radical deprotonates at the C3 hydroxy group. Furthermore, a C2 H-abstraction radical is formed following the thermally induced decay of the trapped electron. The C2 and C3 H-abstraction radicals did not convert into each other. A third radical species formed at low temperatures is a C5 H-abstraction radical. It is unstable above 250 K and decays without any apparent successor. The C2 and C3 H-abstraction radicals are formed thermally and photochemically from the parent trapped electron. The conversions are mediated by hydrogen atoms formed intermediately or by elimination of hydride ions. The thermal decomposition pathway requires further studies, in particular with respect to the possible role of water. Recently, Box et al. analyzed the site of the trapped electron in rhamnose crystals. The present results support the results obtained by these authors (Radiat. Res. 121, 262 (1990)). In particular, trapped electron vs proton distances closely match the conversion mechanisms suggested.     

The structure of the guanine cation: ESR/ENDOR of cyclic guanosine monophosphate single crystals after X irradiation at 10 K.

Following X irradiation of 3',5'-cyclic guanosine monophosphate single crystals at 10 K, several free radicals were trapped and detected by ESR/ENDOR/FSE spectroscopy. The two dominant species both have unpaired spin located on the guanine base. One is the product of net hydrogen atom loss from the exocyclic amino group. The spectroscopic characteristics of this resonance leave this assignment unambiguous. The experimental conditions make it likely that this species was formed by deprotonation of the guanine base cation. The nature of the other species is more uncertain. However, the evidence is consistent with the assignment that it is a net OH adduct to the C4 position of the base. Several species in which the unpaired spin was located on the sugar-phosphate region of the molecule were also observed. The mechanisms for the decay of the primary radicals, also leading to the well-known C8 hydrogen addition radical of the guanine base, are described and discussed.     

Radical formation in X-irradiated single crystals of guanine hydrochloride monohydrate. II. ESR and ENDOR in the range 10-77 K

In a study of guanine.HCl.H2O (Gm) single crystals X-irradiated at temperatures between 10 and 77 K, three radical species were found and characterized by ESR and ENDOR spectroscopy. All three are primary products in that they were present immediately following irradiation at T less than 10 K. Radical I, which apparently can exist in two slightly different conformations, was identified as the product of electron gain by the parent molecule and subsequent protonation at O6. Radical I decayed only after warming the crystals beyond 250 K. Radical II was the guanine cation previously reported (D. M. Close, E. Sagstuen, and W. H. Nelson, J. Chem. Phys. 82, 4386 (1985)); however, ENDOR data are reported here which confirm the previous results. The guanine cation in Gm resulted from electron loss from the parent and subsequent deprotonation at N7. It is proposed that Radical III results from OH attack at C8 of the parent molecule, followed by rupture of the C8-N9 bond and ring opening. The OH radicals thought to produce Radical III result from electron loss by the cocrystallized water molecules. The reaction leading to Radical III, unusual in solid-state radiation chemistry, is thought to be mediated by the specific hydrogen bonding network in this crystal.     

Radiation-induced free radicals in solid 5-halouracil derivatives: single crystals of 5-iododeoxyuridine

X-irradiation of single crystals of 5-iododeoxyuridine (IUdR) in the temperature range 8-300 K produces mainly four different radicals which have been studied by electron spin resonance (e.s.r.) and electron nuclear double resonance (ENDOR)-spectroscopy. At low temperatures, a pi-anion is formed which shows predominantly an interaction of the unpaired electron with a proton at carbon C6 of the base (-11.8 G, -23.9 G, -4.6 G). Above 10-20 K, the anion protonates at C6 to yield a RC-I(CH2)-R' radical comprising alpha-iodo and beta-methylene proton hyperfine interactions. The primary oxidation product is an O5'-situated alkoxy radical RCH2O which shows inequivalent beta-proton couplings of about 100 G and 35 G together with a highly anisotropic g-tensor. Upon warming to 265 K, a C2'-located radical on the deoxyribose is formed which is stable at room temperature. A detailed account of its spectral features as obtained by ENDOR exhibits three different alpha-type couplings, two small beta-protons and a dipolar interaction. Other radicals, not reproducibly observed, involve a C5'-hydroxyalkyl radical and a species related to the base cation at low temperatures.     

Artificial Anti-Tumor Opsonizing Proteins with Fibronectin Scaffolds Engineered for Specificity to Each of the Murine FcγR Types

We have engineered a panel of novel Fn3 scaffold-based proteins that bind with high specificity and affinity to each of the individual mouse Fcγ receptors (mFcγR). These binders were expressed as fusions to anti-tumor antigen single-chain antibodies and mouse serum albumin, creating opsonizing agents that invoke only a single mFcγR response rather than the broader activity of natural Fc isotypes, as well as all previously reported Fc mutants. This panel isolated the capability of each of the four mFcγRs to contribute to macrophage phagocytosis of opsonized tumor cells and in vivo tumor growth control with these monospecific opsonizing fusion proteins. All activating receptors (mFcγRI, mFcγRIII, and mFcγRIV) were capable of driving specific tumor cell phagocytosis to an equivalent extent, while mFcγRII, the inhibitory receptor, did not drive phagocytosis. Monospecific opsonizing fusion proteins that bound mFcγRI alone controlled tumor growth to an extent similar to the most active IgG2a murine isotype. As expected, binding to the inhibitory mFcγRII did not delay tumor growth, but unexpectedly, mFcγRIII also failed to control tumor growth. mFcγRIV exhibited detectable but lesser tumor-growth control leading to less overall survival compared to mFcγRI. Interestingly, in vivo macrophage depletion demonstrates their importance in tumor control with mFcγRIV engagement, but not with mFcγRI. This panel of monospecific mFcγR-binding proteins provides a toolkit for isolating the functional effects of each mFcγR in the context of an intact immune system.     

EPR, ENDOR, and TRIPLE resonance spectroscopy on the neutral flavin radical in Escherichia coli DNA photolyase

Ultraviolet radiation promotes the formation of a cyclobutane ring between adjacent pyrimidine residues on the same DNA strand to form a pyrimidine dimer. Such dimers may be restored to their monomeric forms through the action of a light-absorbing enzyme named DNA photolyase. The redox-active cofactor involved in the light-induced electron transfer reactions of DNA repair and enzyme photoactivation is a noncovalently bound FAD. In this paper, the FAD cofactor of Escherichia coli DNA photolyase was characterized as the neutral flavin semiquinone by EPR spectroscopy at 9.68 and 94.5 GHz. From the high-frequency/high-field EPR spectrum, the principal values of the axially symmetric g-matrix of FADH(*) were extracted. Both EPR spectra show an emerging hyperfine splitting of 0.85 mT that could be assigned to the isotropic hyperfine coupling constant (hfc) of the proton at N(5). To obtain more information about the electron spin density distribution ENDOR and TRIPLE resonance spectroscopies were applied. All major proton hfc's could be measured and unambiguously assigned to molecular positions at the isoalloxazin moiety of FAD. The isotropic hfc's of the protons at C(8alpha) and C(6) are among the smallest values reported for protein-bound neutral flavin semiquinones so far, suggesting a highly restricted delocalization of the unpaired electron spin on the isoalloxazin moiety. Two further hfc's have been detected and assigned to the inequivalent protons at C(1'). Some conclusions about the geometrical arrangement of the ribityl side chain with respect to the isoalloxazin ring could be drawn: Assuming tetrahedral angles at C(1') the dihedral angle between the C(1')-C(2') bond and the 2p(z)() orbital at N(10) has been estimated to be 170.4 degrees +/- 1 degrees.     

ENDOR-assisted study of the stable EPR spectrum of X-irradiated alpha-L-sorbose single crystals: MLCFA and simulation decomposition analyses

After X irradiation of single crystals of alpha-L-sorbose at 295 K, previous electron paramagnetic resonance (EPR), electron nuclear double resonance (ENDOR), and ENDOR-induced EPR (EI-EPR) results have indicated the formation of at least 10 different free radicals, and also that conceivably each carbon in the pyranose ring is a possible radical center. The radicals appear to be formed mostly by net H-abstraction reactions followed by standard elimination (e.g. beta-OH elimination) reactions or proton shifts, in turn leading to ring opening and fragmentation. In the present work, EPR spectra were recorded at room temperature with the external magnetic field along each of the three crystallographic axes subsequent to careful annealing at different temperatures using a high-temperature cavity. Each of the three sets of spectra was subjected to a maximum likelihood common factor analysis (MLCFA) that contributed to a better understanding of the spectral decays. Furthermore, the most stable spectra were simulated by optimization of previous ENDOR and EI-EPR results. The optimized EPR parameters resulted in excellent simulations of the experimental stable sorbose spectra and hence provided an improved insight of their spectral compositions.     

Down-regulation of transforming growth factor-beta receptors: cooperativity between the types I, II, and III receptors and modulation at the cell surface.

The types I, II, and III receptors (RI, RII, RIII) for transforming growth factor-beta (TGF-beta) become down-regulated in response to ligand, presumably via their internalization from the cell surface. This report examines the down-regulation of full-length RI, RII, and RIII in cells endogenously or transiently expressing these receptors. Down-regulation occurred rapidly (within 2 h after TGF-beta1 treatment at 37 degrees C) and showed a dose response, between 10 and 200 pM TGF-beta1, in cells expressing RI, RII, and RIII (Mv1lu and A549 cells). A comparison between Mv1Lu and mutant cell derivatives R-1B (lacking RI) or DR-26 (lacking RII) indicated that all three receptors were necessary for efficient down-regulation. Down-regulation experiments, utilizing TGF-beta-treated 293 cells transiently expressing different combinations of these receptors indicated that neither RII or RIII were down-regulated when expressed alone and that RI was required for maximal down-regulation of RII. RII and RIII were partially down-regulated when these receptors were coexpressed in the absence of RI (in R-1B and 293 cells). Surprisingly, TGF-beta receptors were partially down-regulated in Mv1Lu, A549, and 293 cells treated with TGF-beta1 at 4 degrees C. Microscopic examination of 293 cells coexpressing RI fused to green fluorescent protein (RI-GFP) and RII indicated that, after treatment with TGF-beta1 at 4 degrees C, RI-GFP formed aggregates at the cell surface at this temperature. RI-GFP was not detected at the surface of these cells after TGF-beta1 treatment at 37 degrees C. Our results suggest a two phase mechanism for TGF-beta1 receptor down-regulation involving receptor modulation (aggregation) at the cell surface and internalization.     

Electron paramagnetic resonance and electron nuclear double resonance spectroscopic identification and characterization of the tyrosyl radicals in prostaglandin H synthase 1

The tyrosyl radicals generated in reactions of ethyl hydrogen peroxide with both native and indomethacin-pretreated prostaglandin H synthase 1 (PGHS-1) were examined by low-temperature electron paramagnetic resonance (EPR) and electron nuclear double resonance (ENDOR) spectroscopies. In the reaction of peroxide with the native enzyme at 0 degrees C, the tyrosyl radical EPR signal underwent a continuous reduction in line width and lost intensity as the incubation time increased, changing from an initial, 35-G wide doublet to a wide singlet of slightly smaller line width and finally to a 25-G narrow singlet. The 25-G narrow singlet produced by self-inactivation was distinctly broader than the 22-G narrow singlet obtained by indomethacin treatment. Analysis of the narrow singlet EPR spectra of self-inactivated and indomethacin-pretreated enzymes suggests that they reflect conformationally distinct tyrosyl radicals. ENDOR spectroscopy allowed more detailed characterization by providing hyperfine couplings for ring and methylene protons. These results establish that the wide doublet and the 22-G narrow singlet EPR signals arise from tyrosyl radicals with different side-chain conformations. The wide-singlet ENDOR spectrum, however, is best accounted for as a mixture of native wide-doublet and self-inactivated 25-G narrow-singlet species, consistent with an earlier EPR study [DeGray et al. (1992) J. Biol. Chem. 267, 23583-23588]. We conclude that a tyrosyl residue other than the catalytically essential Y385 species is most likely responsible for the indomethacin-inhibited, narrow-singlet spectrum. Thus, this inhibitor may function by redirecting radical formation to a catalytically inactive side chain. Either radical migration or conformational relaxation at Y385 produces the 25-G narrow singlet during self-inactivation. Our ENDOR data also indicate that the catalytically active, wide-doublet species is not hydrogen bonded, which may enhance its reactivity toward the fatty-acid substrate bound nearby.     

Effect of substrate on the diiron(III) site in stearoyl acyl carrier protein delta 9-desaturase as disclosed by cryoreduction electron paramagnetic resonance/electron nuclear double resonance spectroscopy

The diiron center in stearoyl-acyl carrier protein (ACP) desaturase (DS) from castor plant Ricinus communis catalyzes the dioxygen- and NADPH-dependent introduction of a cis double bond between C9 and C10 of stearoyl-ACP. Radiolytic reduction of diferric DS at 77 K produces an electron paramagnetic resonance (EPR)-detectable mixed-valence center (or [DS(ox)](mv)) that is trapped in the conformation of the diferric precursor and thus provides a sensitive EPR/electron nuclear double resonance (ENDOR) probe of the structure of the diamagnetic diiron(III) state. The cryoreduced DS shows two distinct EPR signals, suggesting the presence of two diiron(III) states: the mu-oxo (major)- and mu-hydroxo (minor)-bridged diiron centers. ENDOR studies show that in the dominant oxo-bridged diferric state each iron(III) coordinates a histidine and a water along with other ligands. Samples containing stoichiometric amounts of stearoyl-ACP show pronounced changes in the EPR and (1)H ENDOR spectra of cryoreduced DS. EPR spectra of the cryoreduced DS-substrate complex reveal two distinct substates of the parent. EPR and ENDOR studies show that both major conformers of the diferric cluster have a mu-oxo bridge. ENDOR shows that the major conformer has a histidine and a water bound to both Fe ions. In the minor conformer, one of the irons has lost the terminal water ligand. The structure of the trapped [DS(ox)](mv) state relaxes upon annealing to 170 K: the mu-oxo bridge in the major cryoreduced DS species protonates on annealing to 170 K; this does not occur for the major DS-substrate complex, even upon annealing to 230 K. The relaxed Fe(II)Fe(III) center in cryoreduced DS and DS-substrate show much less intense and resolved (14)N ENDOR spectra than those of the structurally similar cryoreduced diiron center in ribonucleotide reductase (RNR) protein R2. This difference may reflect some differences in His-Fe bonds. The alterations in the diferric site of DS induced by substrate are suggested to be mediated by conformational changes in the polypeptide chain produced by substrate binding. These structural alterations may provide DS with an additional mechanism for tuning the redox potential of the diferric site. The mixed-valence states of DS are unstable at temperatures above 230 K.