Spin-spin interaction in ethanolamine deaminase

The adenosylcobalamin coenzyme-dependent ethanolamine deaminase from Salmonella typhimurium catalyzes the deamination of aminoethanol to acetaldehyde and ammonia. The radical intermediate observed during steady state turnover of substrate aminoethanol has been characterized by continuous wave electron paramagnetic resonance (EPR) spectroscopy [J. Am. Chem. Soc. 121 (1999) 10522]. This study presents simulations of EPR spectra of this radical intermediate. Quantitative fits to the EPR spectra are achieved with a model of isotropic exchange and magnetic dipolar interaction between the substrate-derived radical and the Co(II) in the corrin ring. The simulated parameters are compared with those of substrate analog 2-aminopropanol-derived radical in the same enzyme. The comparison confirms that the aminoethanol-derived product radical interacts more weakly with the Co(II) than the 2-aminopropanol-derived radical and suggests that the reduction of isotropic exchange between the aminoethanol-derived product radical and the Co(II) is probably due to orientational-dependent wave function overlap. Successful fits to the radical line shapes of different isotope substitutions unequivocally establish that the observed radical intermediate is an pi-electron-based product radical. The derived principal hyperfine values for the 13C(alpha) and 1H(alpha) nucleus are consistent with previous electron nuclear double resonance (ENDOR) studies on similar radicals, thus providing reliable experimental hyperfine coupling constants for comparison with quantum mechanical-based calculations to gain further insight into the molecular structure of the observed radical.     

Radical mechanisms in adenosylmethionine- and adenosylcobalamin-dependent enzymatic reactions

A class of enzymatic reactions of S-adenosylmethionine (AdoMet) has recently been recognized, in which AdoMet plays a novel role by initiating free radical formation through the intermediate formation of 5'-deoxyadenosine-5'-yl, the 5'-deoxyadenosyl radical. The reactions are in this way related to adenosylcobalamin-dependent processes, which also depend on the formation of the 5'-deoxyadenosyl radical as an intermediate. The mechanisms by which the 5'-deoxyadenosyl radical is generated by the AdoMet- and adenosylcobalamin-dependent enzymes are very different. However, the functions of the 5'-deoxyadenosyl radical are similar in that in all cases it abstracts hydrogen from a substrate to form 5'-deoxyadenosine and a substrate-derived free radical. In this paper, the role of the 5'-deoxyadenosyl radical in the reaction of the adenosylcobalamin-dependent reactions will be compared with its role in the AdoMet-dependent reaction of lysine 2,3-aminomutase. The mechanism by which AdoMet is cleaved to the 5'-deoxyadenosyl radical at enzymatic sites will also be discussed.     

Identification of the substrate radical intermediate derived from ethanolamine during catalysis by ethanolamine ammonia-lyase

Rapid-mix freeze-quench (RMFQ) methods and electron paramagnetic resonance (EPR) spectroscopy have been used to characterize the steady-state radical in the deamination of ethanolamine catalyzed by adenosylcobalamin (AdoCbl)-dependent ethanolamine ammonia-lyase (EAL). EPR spectra of the radical intermediates formed with the substrates, [1-13C]ethanolamine, [2-13C]ethanolamine, and unlabeled ethanolamine were acquired using RMFQ trapping methods from 10 ms to completion of the reaction. Resolved 13C hyperfine splitting in EPR spectra of samples prepared with [1-13C]ethanolamine and the absence of such splitting in spectra of samples prepared with [2-13C]ethanolamine show that the unpaired electron is localized on C1 (the carbinol carbon) of the substrate. The 13C splitting from C1 persists from 10 ms throughout the time course of substrate turnover, and there was no evidence of a detectable amount of a product like radical having unpaired spin on C2. These results correct an earlier assignment for this radical intermediate [Warncke, K., et al. (1999) J. Am. Chem. Soc. 121, 10522-10528]. The EPR signals of the substrate radical intermediate are altered by electron spin coupling to the other paramagnetic species, cob(II)alamin, in the active site. The dipole-dipole and exchange interactions as well as the 1-13C hyperfine splitting tensor were analyzed via spectral simulations. The sign of the isotropic exchange interaction indicates a weak ferromagnetic coupling of the two unpaired electrons. A Co2+-radical distance of 8.7 A was obtained from the magnitude of the dipole-dipole interaction. The orientation of the principal axes of the 13C hyperfine splitting tensor shows that the long axis of the spin-bearing p orbital on C1 of the substrate radical makes an angle of approximately 98 degrees with the unique axis of the d(z2) orbital of Co2+.     

ATP-driven electron transfer in enzymatic radical reactions

A novel method to generate organic radicals in enzymatic reactions is described, which is similar to electron transfer in nitrogenase. Component A of 2-hydroxyglutaryl-CoA dehydratase contains a [4Fe-4S] cluster located at the interface between its two identical subunits. The cluster is reduced by one electron derived from ferredoxin or flavodoxin. Hydrolysis of two ATP bound to component A, one to each subunit, enhances the reductive power of the electron and transfers it to component D, the actual dehydratase, where a low potential [4Fe-4S](2+) cluster is probably reduced. Further transfer to the substrate (R)-2-hydroxyglutaryl-CoA probably generates a substrate-derived ketyl radical anion, which expels the adjacent hydroxyl group. The resulting enoxy radical is deprotonated to a product-related ketyl radical anion. Finally the electron is removed by the next incoming substrate leading to the product glutaconyl-CoA and starting a new turnover. A similar, but stoichiometric rather than catalytic electron transfer has been established for the related benzoyl-CoA reductase.     

Probing interactions from solvent-exchangeable protons and monovalent cations with the 1,2-propanediol-1-yl radical intermediate in the reaction of dioldehydrase

The reaction of adenosylcobalamin-dependent dioldehydrase with 1,2-propanediol gives rise to a radical intermediate observable by EPR spectroscopy. This reaction requires a monovalent cation such as potassium ion. The radical signal arises from the formation of a radical pair comprised of the Co(II) of cob(II)alamin and a substrate-related radical generated upon hydrogen abstraction by the 5'-deoxyadenosyl radical. The high-field asymmetric doublet arising from the organic radical has allowed investigation of its composition and environment through the use of EPR spectroscopic techniques. To characterize the protonation state of the oxygen substituents in the radical intermediate, X-band EPR spectroscopy was performed in the presence of D(2)O and compared to the spectrum in H(2)O. Results indicate that the unpaired electron of the steady-state radical couples to a proton on the C(1) hydroxyl group. Other spectroscopic experiments were performed, using either potassium or thallous ion as the activating monovalent cation, in an attempt to exploit the magnetic nature of the (205,203)Tl nucleus to identify any intimate interaction of the radical intermediate with the activating cation. The radical intermediate in complex with dioldehydrase, cob(II)alamin and one of the activating monovalent cations was observed using EPR, ENDOR, and ESEEM spectroscopy. The spectroscopic evidence did not implicate a direct coordination of the activating cation and the substrate derived radical intermediate.     

The substrate radical of Escherichia coli oxygen-independent coproporphyrinogen III oxidase HemN

During porphyrin biosynthesis the oxygen-independent coproporphyrinogen III oxidase (HemN) catalyzes the oxidative decarboxylation of the propionate side chains of rings A and B of coproporphyrinogen III to form protoporphyrinogen IX. The enzyme utilizes a 5'-deoxyadenosyl radical to initiate the decarboxylation reaction, and it has been proposed that this occurs by stereo-specific abstraction of the pro-S-hydrogen atom at the beta-position of the propionate side chains leading to a substrate radical. Here we provide EPR-spectroscopic evidence for intermediacy of the latter radical by observation of an organic radical EPR signal in reduced HemN upon addition of S-adenosyl-L-methionine and the substrate coproporphyrinogen III. This signal (g(av) = 2.0029) shows a complex pattern of well resolved hyperfine splittings from at least five different hydrogen atoms. The radical was characterized using regiospecifically labeled (deuterium or 15N) coproporphyrinogen III molecules. They had been generated from a multienzyme mixture and served as efficient substrates. Reaction of HemN with coproporphyrinogen III, perdeuterated except for the methyl groups, led to the complete loss of resolved proton hyperfine splittings. Substrates in which the hydrogens at both alpha- and beta-positions, or only at the beta-positions of the propionate side chains, or those of the methylene bridges, were deuterated showed that there is coupling with hydrogens at the alpha-, beta-, and methylene bridge positions. Deuterium or 15N labeling of the pyrrole nitrogens without labeling the side chains only led to a slight sharpening of the radical signal. Together, these observations clearly identified the radical signal as substrate-derived and indicated that, upon abstraction of the pro-S-hydrogen atom at the beta-position of the propionate side chain by the 5'-deoxyadenosyl radical, a comparatively stable delocalized substrate radical intermediate is formed in the absence of electron acceptors. The observed hyperfine constants and g values show that this coproporphyrinogenyl radical is allylic and encompasses carbon atoms 3', 3, and 4.     

A substrate radical intermediate in the reaction between ribonucleotide reductase from Escherichia coli and 2'-azido-2'-deoxynucleoside diphosphates

The B2 subunit of ribonucleotide reductase from Escherichia coli contains a tyrosine radical which is essential for enzyme activity. In the reaction between ribonucleotide reductase and the substrate analogue 2'-azido-2'-deoxycytidine 5'-diphosphate a new transient radical is formed. The EPR characteristics of this new radical species are consistent with a localization of the unpaired electron at the sugar moiety of the nucleotide. The radical shows hyperfine couplings to a hydrogen and a nitrogen nucleus, the latter probably being part of the azide substituent. The formation of the nucleotide radical in this suicidal reaction is concomitant with the decay of the tyrosine radical of the B2 subunit. Kinetic data argue for a first (pseudosecond) order decay of the B2 radical via generation of the nucleotide radical followed by a slower first order decay of the nucleotide radical. End products in the reaction are cytosine and radical-free protein B2. In the reaction between bacteriophage T4 ribonucleotide reductase and 2'-azido-2'-deoxycytidine 5'-diphosphate an identical nucleotide radical is formed. The present results are consistent with the hypothesis that the appearance and structure of the transient radical mimic stages in the normal reaction pathway of ribonucleotide reductase, postulated to proceed via 3'-hydrogen abstraction and cation radical formation of the substrate nucleotide (Stubbe, J., and Ackles, D. (1980) J. Biol. Chem. 255, 8027-8030). The nucleotide radical described here might be equivalent to such a cation radical intermediate.     

Secondary isotope effects and structure-reactivity correlations in the dopamine beta-monooxygenase reaction: evidence for a chemical mechanism

The chemical mechanism of hydroxylation, catalyzed by dopamine beta-monooxygenase, has been explored with a combination of secondary kinetic isotope effects and structure-reactivity correlations. Measurement of primary and secondary isotope effects on Vmax/Km under conditions where the intrinsic primary hydrogen isotope effect is known allows calculation of the corresponding intrinsic secondary isotope effect. By this method we have obtained an alpha-deuterium isotope effect, Dk alpha = 1.19 +/- 0.06, with dopamine as substrate. The beta-deuterium isotope effect is indistinguishable from one. The large magnitude of Dk alpha, together with our previous determination of a near maximal primary deuterium isotope effect of 9.4-11, clearly indicates the occurrence of a stepwise process for C-H bond cleavage and C-O bond formation and hence the presence of a substrate-derived intermediate. To probe the nature of this intermediate, a structure-reactivity study was performed by using a series of para-substituted phenylethylamines. Deuterium isotope effects on Vmax and Vmax/Km parameters were determined for all of the substrates, allowing calculation of the rate constants for C-H bond cleavage and product dissociation and dissociation constants for amine and O2 loss from the enzyme-substrate ternary complex. Multiple regression analysis yielded an electronic effect of p = -1.5 for the C-H bond cleavage step, eliminating the possibility of a carbanion intermediate. A negative p value is consistent with formation of either a radical or a carbocation; however, a significantly better correlation is obtained with sigma p rather than sigma p+, implying formation of a radical intermediate via a polarized transition state. Additional effects determined from the regression analyses include steric effects on rate constants for substrate hydroxylation and product release and on KDamine, consistent with a sterically restricted binding site, and a positive electronic effect of p = 1.4 on product dissociation, ascribed to a loss of product from an enzyme-bound Cu(II)-alkoxide complex. These results lead us to propose a mechanism in which O-O homolysis [from a putative Cu(II)-OOH species] and C-H homolysis (from substrate) occur in a concerted fashion, circumventing the formation of a discrete, high energy oxygen species such as hydroxyl radical. The substrate and peroxide-derived radical intermediates thus formed undergo a recombination, kinetically limited by displacement of an intervening water molecule, to give the postulated Cu(II)-alkoxide product complex.     

Localization of a catalytic intermediate bound to the FeMo-cofactor of nitrogenase

Nitrogenase catalyzes the biological reduction of N(2) to ammonia (nitrogen fixation) as well as the reduction of a number of alternative substrates, including acetylene (HC identical with CH) to ethylene (H2C=CH2). It is known that the metallocluster FeMo-cofactor located within the nitrogenase MoFe protein component provides the site of substrate reduction, but the exact site where substrates bind and are reduced on the FeMo-cofactor remains unknown. We have recently shown that the alpha-70 residue of the MoFe protein plays a significant role in defining substrate access to the active site; alpha-70 approaches one face of the FeMo-cofactor, and when valine is substituted by alanine at this position, the substituted nitrogenase is able to accommodate a reduction of the larger alkyne propargyl alcohol (HC identical with CCH(2)OH, propargyl-OH). During this reduction, a substrate-derived intermediate can be trapped on the FeMo-cofactor resulting in an S = 1/2 spin system with a novel electron paramagnetic resonance spectrum. In the present work, trapping of the propargyl-OH-derived or propargyl amine (HC identical with CCH(2)NH(2), propargyl-NH(2))-derived intermediates is shown to be dependent on pH and the presence of histidine at position alpha-195. It is concluded that these catalytic intermediates are stabilized and thereby trapped by H-bonding interactions between either the-OH group or the-NH(3)(+)group and the imidazole epsilon-NH of alpha-195(His). Thus, for the first time it is possible to establish the location of a bound substrate-derived intermediate on the FeMo-cofactor. Refinement of the binding mode and site was accomplished by the use of density functional and force field calculations pointing to an eta(2) coordination at Fe-6 of the FeMo-cofactor.     

Characterization of a succinyl-CoA radical-cob(II)alamin spin triplet intermediate in the reaction catalyzed by adenosylcobalamin-dependent methylmalonyl-CoA mutase

The electron paramagnetic resonance (EPR) spectrum of an intermediate freeze trapped during the steady state of the reaction catalyzed by the adenosylcobalamin (AdoCbl)-dependent enzyme, methylmalonyl-CoA mutase, has been studied. The EPR spectrum is that of a hybrid triplet spin system created as a result of strong electron-electron spin coupling between an organic radical and the low-spin Co(2+) in cob(II)alamin. The spectrum was analyzed by simulation to obtain the zero-field splitting (ZFS) parameters and Euler angles relating the radical-to-cobalt interspin vector to the g axis system of the low-spin Co(2+). Labeling of the substrate with (13)C and (2)H was used to probe the identity of the organic radical partner in the triplet spin system. The patterns of inhomogeneous broadening in the EPR signals produced by [2'-(13)C]methylmalonyl-CoA and [2-(13)C]methylmalonyl-CoA as well as line narrowing resulting from deuterium substitution in the substrate were consistent with those expected for a succinyl-CoA radical wherein the unpaired electron was centered on the carbon alpha to the free carboxyate group of the rearranged radical. The interspin distance and the Euler angles were used to position this product radical into the active site of the enzyme.     

Identification of the 1,2-propanediol-1-yl radical as an intermediate in adenosylcobalamin-dependent diol dehydratase reaction

The reaction catalyzed by adenosylcobalamin-dependent diol dehydratase proceeds by a radical mechanism. A radical pair consisting of the Co(II) of cob(II)alamin and an organic radical intermediate formed during catalysis gives EPR spectra. The high-field doublet and the low-field broad signals arise from the weak interaction of an organic radical with the low-spin Co(II) of cob(II)alamin. To characterize the organic radical intermediate in the diol dehydratase reaction, several deuterated and (13)C-labeled 1,2-propanediols were synthesized, and the EPR spectra observed in the catalysis were measured using them as substrate. The EPR spectra with the substrates deuterated on C1 showed significant line width narrowing of the doublet signal. A distinct change in the hyperfine coupling was seen with [1-(13)C]-1,2-propanediol, but not with the [2-(13)C]-counterpart. Thus, the organic radical intermediate observed by EPR spectroscopy was identified as the 1,2-propanediol-1-yl radical, a C1-centered substrate-derived radical.     

Spin trapping of nitrogen dioxide and of radicals generated from nitrous acid

Spin trapping of nitrogen dioxide radical by several nitrones has been studied. The reaction results in the formation of persistent acyl nitroxides, after the oxidation of the intermediate spin adducts having an -ONO group on C-2 atom. The intermediate is effectively detected when DEPMPO is used as the spin trap. The reaction between PBN or 5,7-di-tert-butyl-3,3-dimethyl indoline N-oxide with nitrous acid gives the corresponding acyl nitroxide only when oxygen is present in the reaction milieu.

On the other hand, nitroso spin traps do not trap ^⋅NO₂ confirming that the unpaired electron of nitrogen dioxide is localized on the oxygen atom.     

The mechanism of adenosylcobalamin-dependent rearrangements

Adenosylcobalamin (AdoCbl)-dependent rearrangements are a group of reactions with no obvious precedents in organic chemistry. In every case, they are characterized by a mechanism in which a hydrogen atom on one carbon atom exchanges places with a group X on an adjacent carbon: (formula; see text) Much experimental work indicates that an AdoCbl rearrangement is initiated by homolysis of the C-Co bond of the cofactor. The migrating hydrogen is then abstracted from the substrate by the resulting 5'-deoxyadenosyl radical, or by a second radical that is generated elsewhere at the active site, and, after the migration of group X, is returned to the product in a similar reaction. In at least some of the rearrangements, group X migration may occur via a cation radical intermediate that formed by the departure of X with its electrons, a process assisted by the unpaired electron left behind on the adjacent carbon after the abstraction of the migrating hydrogen. Once C-Co bond cleavage has initiated the reaction by producing a free radical at the active site, the corrin ring plays no further role in the rearrangements.     

Molecular dynamics simulations of arachidonic acid-derived pentadienyl radical intermediate complexes with COX-1 and COX-2: insights into oxygenation regio- and stereoselectivity

The two cyclooxygenase enzymes, COX-1 and COX-2, are responsible for the committed step in prostaglandin biosynthesis and are the targets of the nonsteroidal antiinflammatory drugs aspirin and ibuprofen and the COX-2 selective inhibitors, Celebrex, Vioxx, and Bextra. The enzymes are remarkable in that they catalyze two dioxygenations and two cyclizations of the native substrate, arachidonic acid, with near absolute regio- and stereoselectivity. Several theories have been advanced to explain the nature of enzymatic control over this series of reactions, including suggestions of steric shielding and oxygen channeling. As proposed here, selective radical trapping and spin localization in the substrate-derived pentadienyl radical intermediate can also be envisioned. Herein we describe the results of explicit, 10 ns molecular dynamics simulations of both COX-1 and COX-2 with the substrate-derived pentadienyl radical intermediate bound in the active site. The enzymes' influence on the conformation of the pentadienyl radical was investigated, along with the accessible space above and below the radical plane and the width of several channels to the active site that could function as access routes for molecular oxygen. Additional simulations demonstrated the extent of molecular oxygen mobility within the active site. The results suggest that spin localization is unlikely to play a role in enzymatic control of this reaction. Instead, a combination of oxygen channeling, steric shielding, and selective radical trapping appears to be responsible. This work adds a dynamic perspective to the strong foundation of static structural data available for these enzymes.     

Analysis of the electron paramagnetic resonance spectrum of a radical intermediate in the coenzyme B(12)-dependent ethanolamine ammonia-lyase catalyzed reaction of S-2-aminopropanol

The structure of the steady-state radical intermediate in the deamination of S-2-aminopropanol catalyzed by ethanolamine ammonia-lyase (EAL) from Salmonella typhimurium has been probed by electron paramagnetic resonance (EPR) spectroscopy using isotopically labeled forms of the substrate and of the adenosylcobalamin cofactor. Electron spin-spin coupling between the radical, centered on the carbon skeleton of the substrate, and the low-spin Co(2+) in cob(II)alamin (B(12r)) produces a dominant splitting of the EPR signals of both the radical and the Co(2+). Analysis of the exchange and dipole-dipole contributions to the spin-spin coupling indicates that the two paramagnetic centers are separated by approximately 11 A. Experiments with (13)C- and with (2)H-labeled forms of S-2-aminopropanol show that the radical is centered on C1 of the carbon skeleton of the substrate in agreement with an earlier report [Babior, B. M., Moss, T. H., Orme-Johnson, W. H., and Beinert, H., (1974) J. Biol. Chem. 249, 4537-4544]. Experiments with perdeutero-S-2-aminopropanol and [2-(15)N]-perdeutero-S-2-aminopropanol reveal a strong hyperfine splitting from the substrate nitrogen, which indicates that the radical is the initial substrate radical created by abstraction of a hydrogen atom from C1 of S-2-aminopropanol. The strong nitrogen hyperfine splitting further indicates that the amino substituent at C2 is approximately eclipsed with respect to the half-occupied p orbital at C1. Experiments with adenosylcobalamin enriched in (15)N in the dimethylbenzimidazole moiety show that the axial base of the cofactor remains attached to the Co(2+) in a functional steady-state reaction intermediate.     

Characterization of an allylic analogue of the 5'-deoxyadenosyl radical: an intermediate in the reaction of lysine 2,3-aminomutase.

An allylic analogue of the 5'-deoxyadenosyl radical has been characterized at the active site of lysine 2,3-aminomutase (LAM) by electron paramagnetic resonance (EPR) spectroscopy. The anhydroadenosyl radical, 5'-deoxy-3',4'-anhydroadenosine-5'-yl, is a surrogate of the less stable 5'-deoxyadenosyl radical, which has never been observed but has been postulated to be a radical intermediate in the catalytic cycles of a number of enzymes. An earlier communication [Magnusson, O.Th., Reed, G. H., and Frey, P. A. (1999) J. Am. Chem. Soc. 121, 9764-9765] included the initial spectroscopic identification at 77 K of the radical, which is formed upon replacement of S-adenosylmethionine by S-3',4'-anhydroadenosylmethionine as a coenzyme for LAM. The electron paramagnetic resonance spectrum of the radical changes dramatically between 77 and 4.5 K. This unusual temperature dependence is attributed to a spin-spin interaction between the radical and thermally populated, higher spin states of the [4Fe-4S]+2 center, which is diamagnetic at 4.5 K. The EPR spectra of the radical at 4.5 K have been analyzed using isotopic substitutions and simulations. Analysis of the nuclear hyperfine splitting shows that the unpaired spin is distributed equally between C5'- and C3'- as expected for an allylic radical. Hyperfine splitting from the beta-proton at C-2'(H) shows that the dihedral angle to the p(z)-orbital at C-3' is approximately 37 degrees. This conformation is in good agreement with a structural model of the radical. The rate of formation of the allylic radical shows that it is kinetically competent as an intermediate. Measurements of 2H kinetic isotope effects indicate that with lysine as the substrate, the rate-limiting steps follow initial reductive cleavage of the coenzyme analogue.     

Identification of a new reaction intermediate in the oxidation of methylamine dehydrogenase by amicyanin

The two-electron oxidation of tryptophan tryptophylquinone (TTQ) in substrate-reduced methylamine dehydrogenase (MADH) by amicyanin is known to proceed via an N-semiquinone intermediate in which the substrate-derived amino group remains covalently attached to TTQ [Bishop, G. R., and Davidson, V. L. (1996) Biochemistry 35, 8948-8954]. A new method for the stoichiometric formation of the N-semiquinone in vitro has allowed the study of the oxidation of the N-semiquinone by amicyanin in greater detail than was previously possible. Conversion of N-semiquinone TTQ to the quinone requires two biochemical events, electron transfer to amicyanin and release of ammonia from TTQ. Using rapid-scanning stopped-flow spectroscopy, it is shown that this occurs by a sequential mechanism in which oxidation to an imine (N-quinone) precedes hydrolysis by water and ammonia release. Under certain reaction conditions, the N-quinone intermediate accumulates prior to the relatively slow hydrolysis step. Correlation of these transient kinetic data with steady-state kinetic data indicates that the slow hydrolysis of the N-quinone by water does not occur in the steady state. In the presence of excess substrate, the next methylamine molecule initiates a nucleophilic attack of the N-quinone TTQ, causing release of ammonia that is concomitant with the formation of the next enzyme-substrate cofactor adduct. In light of these results, the usually accepted steady-state reaction mechanism of MADH is revised and clarified to indicate that reactions of the quinone form of TTQ are side reactions of the normal catalytic pathway. The relevance of these conclusions to the reaction mechanisms of other enzymes with carbonyl cofactors, the reactions of which proceed via Schiff base intermediates, is also discussed.     

Mechanism of p21Ras S-nitrosylation and kinetics of nitric oxide-mediated guanine nucleotide exchange

Nitric oxide (NO), a highly reactive redox molecule, can react with protein thiols and protein metal centers to regulate a multitude of physiological processes. NO has been shown to promote guanine nucleotide exchange on the critical cellular signaling protein p21Ras (Ras) by S-nitrosylation of a redox-active thiol group (Cys(118)). This increases cellular Ras-GTP levels in vivo, leading to activation of downstream signaling pathways. Yet the process by which this occurs is not clear. Although several feasible mechanisms for protein S-nitrosylation with NO and NO donating have been proposed, results obtained from our studies suggest that Ras can be S-nitrosylated by direct reaction of Cys(118) with nitrogen dioxide (*NO(2)), a reaction product of NO with O(2), via a Ras thiyl-radical intermediate (Ras-S*). Results from our studies also indicate that Ras Cys(118) can be S-nitrosylated by direct reaction of Cys(118) with a glutathionyl radical (GS*), a reaction product derived from homolytic cleavage of S-nitrosoglutathione (GSNO). Moreover, we present evidence that reaction of GS* with Ras generates a Ras-S* intermediate during GSNO-mediated Ras S-nitrosylation. The Ras-S(*) radical intermediate formed from reaction of the Ras thiol with either *NO(2) or GS*, in turn, reacts with NO to complete Ras S-nitrosylation. NO and GSNO modulate Ras activity by promoting guanine nucleotide dissociation from Ras. Our results suggest that formation of the Ras radical intermediate, Ras-S*, may perturb interactions between Ras and its guanine nucleotide substrate, resulting in enhancement of guanine nucleotide dissociation from Ras.     

Electron spin echo envelope modulation spectroscopy in photosystem I.

The applications of electron spin echo envelope modulation (ESEEM) spectroscopy to study paramagnetic centers in photosystem I (PSI) are reviewed with special attention to the novel spectroscopic techniques applied and the structural information obtained. We briefly summarize the physical principles and experimental techniques of ESEEM, the spectral shapes and the methods for their analysis. In PSI, ESEEM spectroscopy has been used to the study of the cation radical form of the primary electron donor chlorophyll species, P(700)(+), and the phyllosemiquinone anion radical, A(1)(-), that acts as a low-potential electron carrier. For P(700)(+), ESEEM has contributed to a debate concerning whether the cation is localized on a one or two chlorophyll molecules. This debate is treated in detail and relevant data from other methods, particularly electron nuclear double resonance (ENDOR), are also discussed. It is concluded that the ESEEM and ENDOR data can be explained in terms of five distinct nitrogen couplings, four from the tetrapyrrole ring and a fifth from an axial ligand. Thus the ENDOR and ESEEM data can be fully accounted for based on the spin density being localized on a single chlorophyll molecule. This does not eliminate the possibility that some of the unpaired spin is shared with the other chlorophyll of P(700)(+); so far, however, no unambiguous evidence has been obtained from these electron paramagnetic resonance methods. The ESEEM of the phyllosemiquinone radical A(1)(-) provided the first evidence for a tryptophan molecule pi-stacked over the semiquinone and for a weaker interaction from an additional nitrogen nucleus. Recent site-directed mutagenesis studies verified the presence of the tryptophan close to A(1), while the recent crystal structure showed that the tryptophan was indeed pi-stacked and that a weak potential H-bond from an amide backbone to one of the (semi)quinone carbonyls is probably the origin of the to the second nitrogen coupling seen in the ESEEM. ESEEM has already played an important role in the structural characterization on PSI and since it specifically probes the radical forms of the chromophores and their protein environment, the information obtained is complimentary to the crystallography. ESEEM then will continue to provide structural information that is often unavailable using other methods.     

ENDOR structural characterization of a catalytically competent acylenzyme reaction intermediate of wild-type TEM-1 beta-lactamase confirms glutamate-166 as the base catalyst

The catalytically competent active-site structure of a true acylenzyme reaction intermediate of TEM-1 beta-lactamase formed with the kinetically specific spin-labeled substrate 6-N-(2,2,5,5-tetramethyl-1-oxypyrrolinyl-3-carboxyl)-penicillanic acid isolated under cryoenzymologic conditions has been determined by angle-selected electron nuclear double resonance (ENDOR) spectroscopy. Cryoenzymologic experiments with use of the chromophoric substrate 6-N-[3-(2-furanyl)-propen-2-oyl]-penicillanic acid showed that the acylenzyme reaction intermediate could be stabilized in the -35 to -75 degrees C range with a half-life suitably long to allow freeze-quenching of the reaction species for ENDOR studies while a noncovalent Michaelis complex could be optically identified at temperatures only below -70 degrees C. The wild-type, Glu166Asn, Glu240Cys, and Met272Cys mutant forms of the mature enzyme were overexpressed in perdeuterated minimal medium to allow detection and assignment of proton resonances specific for the substrate and chemically modified amino acid residues in the active site. From analysis of the dependence of the ENDOR spectra on the setting of the static laboratory magnetic field H0, the dipolar contributions to the principal hyperfine coupling components were estimated to calculate the separations between the unpaired electron of the nitroxyl group and isotopically identified nuclei. These electron-nucleus distances were applied as constraints to assign the conformation of the substrate in the active site and of amino acid side chains by molecular modeling. Of special interest was that the ENDOR spectra revealed a water molecule sequestered in the active site of the acylenzyme of the wild-type protein that was not detected in the deacylation impaired Glu166Asn mutant. On the basis of the X-ray structure of the enzyme, the ENDOR distance constraints placed this water molecule within hydrogen-bonding distance to the carboxylate side chain of glutamate-166 as if it were poised for nucleophilic attack of the scissile ester bond. The ENDOR results provide experimental evidence of glutamate-166 in its functional role as the general base catalyst in the wild-type enzyme for hydrolytic breakdown of the acylenzyme reaction intermediate of TEM-1 beta-lactamase.