Biomedical subjects

Brian M Hoffman

Publications and source records attributed to Brian M Hoffman.

At least 37 records · Page 2Linked to original sources

PubMed2005

Equilibrium/nonequilibrium initial configurations in forward/reverse electron transfer within mixed-metal hemoglobin hybrids.

In a protein-protein electron transfer (ET) photocycle, the "forward" ET reaction is initiated with the excited complex, [3DA], in an equilibrium ensemble of configurations, the majority of which exhibit less than the maximal ET matrix element. In contrast, the charge-separated intermediate complex is formed in a nonequilibrium set of configurations with maximal ET matrix elements and would be expected to return to the ground state with the largest rate constant possible unless conformational interconversion first "breaks the connection" and the complex converts to less-reactive substates. According to this analysis, the forward and back ET reactions should show a differential response to viscosity, and the latter could even show an increased rate constant under conditions which suppress departure from the reactive configuration(s). We now report that the viscosity dependences of forward and back ET rate constants for the photocycle within the [alpha2(Zn),beta2(Fe3+N3-)] mixed-metal hemoglobin hybrid at pH 7 show the anticipated behavior: kf decreases as viscosity increases, but, in sharp contrast, kb increases strongly.

Ferric Compounds↗

PubMed2005

Electron inventory, kinetic assignment (E(n)), structure, and bonding of nitrogenase turnover intermediates with C2H2 and CO.

Improved 1H ENDOR data from the S(EPR1) intermediate formed during turnover of the nitrogenase alpha-195Gln MoFe protein with C2(1,2)H2 in (1,2)H2O buffers, taken in context with the recent study of the intermediate formed from propargyl alcohol, indicate that S(EPR1) is a product complex, likely with C2H4 bound as a ferracycle to a single Fe of the FeMo-cofactor active site. 35 GHz CW and Mims pulsed 57Fe ENDOR of 57Fe-enriched S(EPR1) cofactor indicates that it exhibits the same valencies as those of the CO-bound cofactor of the lo-CO intermediate formed during turnover with CO, [Mo4+, Fe3+, Fe6(2+), S9(2-)(d43)](+1), reduced by m = 2 electrons relative to the resting-state cofactor. Consideration of 57Fe hyperfine coupling in S(EPR1) and lo-CO leads to a picture in which CO bridges two Fe of lo-CO, while the C2H4 of S(EPR1) binds to one of these. To correlate these and other intermediates with Lowe-Thorneley (LT) kinetic schemes for substrate reduction, we introduce the concept of an "electron inventory". It partitions the number of electrons a MoFe protein intermediate has accepted from the Fe protein (n) into the number transmitted to the substrate (s), the number that remain on the intermediate cofactor (m), and the additional number delivered to the cofactor from the P clusters (p): n = m + s - p (with p = 0 here). The cofactors of lo-CO and S(EPR1) both are reduced by m = 2 electrons, but the intermediates are not at the same LT reduction stage (E(n)): (n = 2; m = 2, s = 0) for lo-CO; (n = 4; s = 2, m = 2) for S(EPR1). This is the first proposed correlation of an LT E(n) kinetic state with a well-defined chemical state of the enzyme.

Acetylene↗

PubMed2005

Novel metal-linked face-to-face porphyrazine dimer.

We report the synthesis and physical studies of a novel porphyrazine (pz) dimer [1[Ni,Cu]]2, which has Ni(II) ions incorporated into the pz cores and is linked by two Cu(II) ions coordinated to bis(picolinamide) chelates attached to the pz periphery. [1[Ni,Cu]]2 was prepared from precursor pz 2 with a selenodiazole ring fused to the pz core. This ring was deprotected to form the diamino-pz 3, which reacted with 2 mol of picolinoyl chloride hydrochloride to form pz 1[2H,2H], with peripheral bis(picolinamide) chelates; this was metalated to form [1[Ni,Cu]]2. The crystal structures of 1[2H,2H] and [1[Ni,Cu]]2 are presented. The latter is a dimer in which parallel, face-to-face pz's with an average separation of 3.30 angstroms are linked through the peripheral picolinamide ligands by a pair of peripheral Cu(II) ions. Each Cu(II) is coordinated with distorted square-planar geometry by a picolinamide from each pz. In this report, we focus on the interaction of these two peripheral Cu(II) ions. We discuss the preparation and magnetic properties of the pz dimer complex [1[Ni,Cu]]2 with two Cu(II) ions in the peripheral chelate but a diamagnetic metal ion Ni(II) in the pz core. Although [1[Ni,Cu]]2 contains two Cu(II) ions (S = 1/2), we could detect no electron paramagnetic resonance signal, which suggests very strong antiferromagnetic exchange between those two Cu(II) ions. Temperature-dependent magnetic susceptibility measurement gives an exchange splitting between the S = 0 ground state and the excited triplet state of delta = 660 cm(-1).

Journal Article↗

PubMed2005

Intermediates trapped during nitrogenase reduction of N triple bond N, CH3-N=NH, and H2N-NH2.

A high population intermediate has been trapped on the nitrogenase active site FeMo cofactor during reduction of N2. In addition, intermediates have been trapped during reduction of CH3-N=NH by the alpha-195Gln variant and during reduction of H2N-NH2 by the alpha-70Ala/alpha-195Gln variant. Each of these trapped states shows an EPR signal arising from an S = 1/2 state of the FeMo cofactor. 15N ENDOR shows that each intermediate has a nitrogenous species bound to the FeMo cofactor, with a single type of N seen for each bound intermediate. The g tensors are unique to each intermediate, g(e) = [2.084, 1.993, 1.969], g(m) = [2.083, 2.021, 1.993], g(l) = [2.082, 2.015, 1.987], as are the 15N hyperfine couplings at g1, which suggests that three distinct stages of NN reduction may have been trapped. The 1H ENDOR spectra show that the N2 intermediate is at a distinct and earlier stage of reduction from the other two, so at least two stages of NN reduction have been trapped. Some possible structures of the hydrazine intermediate are presented.

Amides↗

PubMed2005

Cryoreduction EPR and 13C, 19F ENDOR study of substrate-bound substates and solvent kinetic isotope effects in the catalytic cycle of cytochrome P450cam and its T252A mutant.

We recently used cryoreduction EPR/ENDOR techniques to show that a substrate can modulate the properties of both the monooxygenase active-oxygen intermediates and of the proton-delivery network which encompasses them. In the present report we use Q-band pulsed 19F ENDOR (Mims 3-pulse sequence) to examine the substrate binding geometries of camphor, through use of the 5,5'--difluorocamphor, and 13C ENDOR to examine the binding of 5-methylenyl camphor labeled with 13C at C11. These probes are examined in multiple states of the catalytic cycle of P450cam and its T252A mutant. As part of this investigation we further report a new cryoreduction reaction, the reduction of a ferroheme to the EPR-visible Fe(I) state, and use it to probe the substrate binding to the EPR-silent ferroheme state. Finally we report the solvent kinetic isotope effect on the decay of the camphor complex of the hydroperoxo-ferric intermediate, the first such measurement on an individual step within the P450cam reaction cycle. Following reduction of oxyferrous-P450cam, this step is the rate-limiting step in camphor hydroxylation, and its solv-KIE of 1.8 at 190 K establishes that it involves activation of the hydroperoxo moiety by transfer of the 'second' proton of catalysis. We suggest that the finding that the heme pocket can exist in multiple substates, including multiple substrate binding locations, even in P450cam, along with the established possibility that the hydroperoxo-ferriheme intermediate can react with substrate, may explain the formation of multiple products by P450s.

Alanine↗

PubMed2005

The interstitial atom of the nitrogenase FeMo-cofactor: ENDOR and ESEEM evidence that it is not a nitrogen.

X-ray crystallographic study of the nitrogenase MoFe protein revealed electron density from an atom (denoted X) inside the active-site metal cluster, the [MoFe7S9:homocitrate] FeMo-cofactor. The electron density associated with X is consistent with a single N, O, or C atom. We now have tested whether X is an N or not by comparing the Q-band ENDOR and ESEEM signals from resting-state (S = 3/2) MoFe protein and NMF-extracted FeMo-co from bacteria grown with either 14N or 15N as the exclusive N source. All of the 14N or 15N signals associated with the protein are lost upon extraction of the FeMo-co. We interpret this as strong evidence that X is not an N.

Crystallography, X-Ray↗

PubMed2005

Synthesis and magnetic properties comparison of M-Cu(II) and M-VO(II) Schiff base-porphyrazine complexes: what is the mechanism for spin-coupling?

Dimetallic Schiff base-porphyrazine (pz) compounds, denoted 1[M(1); M(2); R], have been prepared, where metal ion M(1) is incorporated into the pz core, and metal ion M(2) is bound to a bis(5-tert-butylsalicylidenimine) chelate built onto two amino nitrogens attached to the pz periphery; R is a solubilizing group (either propyl (Pr) or 3,4,5-trimethoxyphenyl (TMP)) attached to the remaining carbons of the pz periphery. The synthesis of 1[Cu; Cu; R], 1[Cu; VO; R], 1[ClMn; Cu; Pr], and 1[ClMn; VO; Pr] is discussed, the crystal structures of 1[Cu; Cu; TMP] and 1[ClMn; VO; Pr] are presented, and the magnetic properties of these compounds are compared. The pattern of ligand-mediated exchange coupling in these complexes is startling: for the Cu-M(2) complexes 1[Cu; VO; R] and 1[Cu; Cu; R], 2 x 10(2) < or = |J(Cu-VO)/J(Cu-Cu)|; for the ClMn-M(2) complexes 1[ClMn; Cu; Pr] and 1[ClMn; VO; Pr], J(ClMn-VO)/J(ClMn-Cu) approximately 1/3, an inverse ratio from that of the Cu-M(2) complexes, but with lesser discrimination. This coupling pattern is explained in terms of a novel orientation relative to the M(1)-M(2) direction: the "square-planar" Schiff base ligand set of M(2) is rotated in-plane by 45 degrees relative to the effectively coplanar pz ligand set of M(1).

Journal Article↗

PubMed2005

ROM polymerization-capture-release: application to the synthesis of unsymmetrical porphyrazinedithiols and peripherally metalated derivatives.

Crossover Linstead macrocyclization of a doubly norbornenyl-functionalized dimercaptomaleonitrile with dipropylmaleonitrile gave a crude mixture of porphyrazines containing the hexapropylporphyrazinedithiol magnesium complex. The mixture was subjected to ring-opening metathesis polymerization to yield the insoluble porphyrazinedithiol-functionalized polymers. Cleavage from the polymer backbone using mercury(II) acetate followed by reaction with electrophiles gave access to a range of thioporphyrazinedithiol derivatives including solitaire porphyrazines. Studies into the possible uses of hexapropyl-2,3-di-(carboxymethylthio)porphyrazine in sensing metal cations in solution are described.

Journal Article↗

PubMed2005

Enzyme control of small-molecule coordination in FosA as revealed by 31P pulsed ENDOR and ESE-EPR.

FosA is a manganese metalloglutathione transferase that confers resistance to the broad-spectrum antibiotic fosfomycin, which contains a phosphonate group. The active site of this enzyme consists of a high-spin Mn(2+) ion coordinated by endogenous ligands (a glutamate and two histidine residues) and by exogenous ligands, such as substrate fosfomycin. To study the Mn(2+) coordination environment of FosA in the presence of substrate and the inhibitors phosphonoformate and phosphate, we have used (31)P pulsed electron-nuclear double resonance (ENDOR) at 35 GHz to obtain metrical information from (31)P-Mn(2+) interactions. We have found that continuous wave (CW) (31)P ENDOR is not successful in the study of phosphates and phosphonates coordinated to Mn(2+). Parallel studies of phosph(on)ate binding to the Mn(2+) of FosA and to aqueous Mn(2+) ion disclose how the enzyme modifies the coordination of these molecules to the active site Mn(2+). Through analysis of (31)P hyperfine parameters derived from simulations of the ENDOR spectra we have determined the binding modes of the phosph(on)ates in each sample and discerned details of the geometric and electronic structure of the metal center. The (31)P ENDOR studies of the protein samples agree with, or improve on, the Mn-P distances determined from crystal structures and provide Mn-phosph(on)ate bonding information not available from these studies. Electron spin echo electron paramagnetic resonance (ESE-EPR) spectra have also been recorded. Simulation of these spectra yield the axial and rhombic components of the Mn(2+) (S = (5)/(2)) zero-field splitting (zfs) tensor. Comparison of structural inferences based on these zfs parameters both with the known enzyme structures and the (31)P ENDOR results establishes that the time-honored procedure of analyzing Mn(2+) zfs parameters to describe the coordination environment of the metal ion is not valid or productive.

Bacterial Proteins↗

PubMed2005

Trapping a hydrazine reduction intermediate on the nitrogenase active site.

A major challenge in understanding the mechanism of nitrogenase, the enzyme responsible for the biological fixation of N(2) to two ammonias, is to trap a nitrogenous substrate at the enzyme active site in a state that is amenable to further characterization. In the present work, a strategy is described that results in the trapping of the substrate hydrazine (H(2)N-NH(2)) as an adduct bound to the active site metal cluster of nitrogenase, and this bound adduct is characterized by EPR and ENDOR spectroscopies. Earlier work has been interpreted to indicate that nitrogenous (e.g., N(2) and hydrazine) as well as alkyne (e.g., acetylene) substrates can bind at a common FeS face of the FeMo-cofactor composed of Fe atoms 2, 3, 6, and 7. Substitution of alpha-70(Val) that resides over this FeS face by the smaller amino acid alanine was also previously shown to improve the affinity and reduction rate for hydrazine. We now show that when alpha-195(His), a putative proton donor near the active site, is substituted by glutamine in combination with substitution of alpha-70(Val) by alanine, and the resulting doubly substituted MoFe protein (alpha-70(Ala)/alpha-195(Gln)) is turned over with hydrazine as substrate, the FeMo-cofactor can be freeze-trapped in a S = (1)/(2) state in high yield ( approximately 70%). The presumed hydrazine-FeMo-cofactor adduct displays a rhombic EPR signal with g = [2.09, 2.01, 1.93]. The optimal pH for the population of this state was found to be 7.4. The EPR signal showed a Curie law temperature dependence similar to the resting state EPR signal. Mims pulsed ENDOR spectroscopy at 35 GHz using (15)N-labeled hydrazine reveals that the trapped intermediate incorporates a hydrazine-derived species bound to the FeMo-cofactor; in spectra taken at g(1) this species gives a single observed (15)N signal, A(g(1)) = 1.5 MHz.

Azotobacter vinelandii↗

PubMed2005

ENDOR studies of the ligation and structure of the non-heme iron site in ACC oxidase.

Ethylene is a plant hormone involved in all stages of growth and development, including regulation of germination, responses to environmental stress, and fruit ripening. The final step in ethylene biosynthesis, oxidation of 1-aminocyclopropane-1-carboxylic acid (ACC) to yield ethylene, is catalyzed by ACC oxidase (ACCO). In a previous EPR and ENDOR study of the EPR-active Fe(II)-nitrosyl, [FeNO],(7) complex of ACCO, we demonstrated that both the amino and the carboxyl moieties of the inhibitor d,l-alanine, and the substrate ACC by analogy, coordinate to the Fe(II) ion in the Fe(II)-NO-ACC ternary complex. In this report, we use 35 GHz pulsed and CW ENDOR spectroscopy to examine the coordination of Fe by ACCO in more detail. ENDOR data for selectively (15)N-labeled derivatives of substrate-free ACCO-NO (E-NO) and substrate/inhibitor-bound ACCO-NO (E-NO-S) have identified two histidines as protein-derived ligands to Fe; (1,2)H and (17)O ENDOR of samples in D(2)O and H(2)(17)O solvent have confirmed the presence of water in the substrate-free Fe(II) coordination sphere (E-NO). Analysis of orientation-selective (14,15)N and (17)O ENDOR data is interpreted in terms of a structural model of the ACCO active site, both in the presence (E-NO-S) and in the absence (E-NO) of substrate. Evidence is also given that substrate binding dictates the orientation of bound O(2).

Amino Acid Oxidoreductases↗

PubMed2005

Trapping H- bound to the nitrogenase FeMo-cofactor active site during H2 evolution: characterization by ENDOR spectroscopy.

We here show that the iron-molybdenum (FeMo)-cofactor of the nitrogenase alpha-70(Ile) molybdenum-iron (MoFe) protein variant accumulates a novel S = (1)/(2) state that can be trapped during the reduction of protons to H(2). (1,2)H-ENDOR measurements disclose the presence of two protons/hydrides (H(+/)(-)) whose hyperfine tensors have been determined from two-dimensional field-frequency (1)H ENDOR plots. The two H(+/)(-) have large isotropic hyperfine couplings, A(iso)( )() approximately 23 MHz, which shows they are bound to the cofactor. The favored analysis for these plots indicates that the two H(+/)(-) have the same principal values, which indicates that they are chemically equivalent. The tensors are further related to each other by a permutation of the tensor components, which indicates an underlying symmetry of binding relative to the cofactor. At present, no model for the structure of the iron-molybdenum (FeMo)-cofactor in the S = (1)/(2) state trapped during the reduction of H(+) can be shown unequivocally to satisfy all of the constraints generated by the ENDOR analysis. The data disfavors any model that involves protonation of sulfides, and thus suggests that the intermediate instead contains two chemically equivalent bound hydrides; it appears unlikely that these are terminal monohydrides.

Binding Sites↗

PubMed2005

Hopping in the electron-transfer photocycle of the 1:1 complex of Zn-cytochrome c peroxidase with cytochrome c.

The physiological electron-transfer (ET) partners, cytochrome c peroxidase (CcP) and cytochrome c (Cc)1, can be modified to exhibit photoinitiated ET through substitution of Zn (or Mg) for Fe in either partner. Laser excitation of the Zn-porphyrin (ZnP) to its triplet excited state (3ZnP) initiates direct heme-heme ET to the ferriheme center of its partner across the protein-protein interface. This photoinitiated ET produces the charge-separated intermediate, I = [ZnP+CcP, Fe2+Cc], with a metalloporphyrin pi-cation radical (ZnP+) in the donor protein and a ferroheme acceptor protein. I, in general, is thought to return to the ground state by a thermal ET process that involves direct heme-heme back-ET to complete a simple photocycle. We here contrast intracomplex ET between yeast iso-1 Cc and ZnCcP(WT) (wild-type) with that for two ZnCcP(X) variants: X = W191F, with redox-active W191 replaced by Phe; WYM4, a W191F mutant with further replacement of four other potentially redox-active sites (W51F, Y187F, Y229F, and Y236F). The results show that W191 acts as an ET mediator, which "short-circuits" the direct heme-heme back-ET through a two-step, hopping process in which the ZnP+ cation radical formed by photoinitiated ET rapidly oxidizes W191, and the resultant W191+, in turn, rapidly oxidizes Fe2+Cc.

Cytochrome-c Peroxidase↗

PubMed2005

ROM polymerization-capture-release strategy for the chromatography-free synthesis of novel unsymmetrical porphyrazines.

[reaction: see text] Crossover-Linstead macrocyclization reactions of two norbornenyl-tagged diaminomaleonitriles with dipropylmaleonitrile gave access to crude mixtures of porphyrazines containing diamino-hexapropyl-porphyrazine magnesium complexes. The mixtures were subjected to ring-opening metathesis polymerization to yield the insoluble diaminoporphyrazine-functionalized polymers. Acid-mediated cleavage from the polymer backbone followed by acylation of the resultant sensitive macrocyclic diamines gave monoacetyl-, monotrifluoroacetyl-, and ditrifluoroacetyl-substituted porphyrazinediamines. Conversion of these amido-porphyrazines to the corresponding zinc macrocycles and studies of their electronic absorption and emission spectra, electrochemistry, and photophysics are described.

Journal Article↗

PubMed2005

Differential influence of dynamic processes on forward and reverse electron transfer across a protein-protein interface.

We propose that the forward and reverse halves of a flash-induced protein-protein electron transfer (ET) photocycle should exhibit differential responses to dynamic interconversion of configurations when the most stable configuration is not the most reactive, because the reactants exist in different initial configurations: the flash-photoinitiated forward ET process begins with the protein partners in an equilibrium ensemble of configurations, many of which have little or no reactivity, whereas the reactant of the thermal back ET (the charge-separated intermediate) is formed in a nonequilibrium, "activated" protein configuration. We report evidence for this proposal in measurements on (i) mixed-metal hemoglobin hybrids, (ii) the complex between cytochrome c peroxidase and cytochrome c, and (iii and iv) the complexes of myoglobin and isolated hemoglobin alpha-chains with cytochrome b(5). For all three systems, forward and reverse ET does respond differently to modulation of dynamic processes; further, the response to changes in viscosity is different for each system.

Cytochrome-c Peroxidase↗

PubMed2005

Spectroscopic approaches to elucidating novel iron-sulfur chemistry in the "radical-Sam" protein superfamily.

Electron paramagnetic resonance (EPR), electron-nuclear double resonance (ENDOR), and Mössbauer spectroscopies and other physical methods have provided important new insights into the radical-SAM superfamily of proteins, which use iron-sulfur clusters and S-adenosylmethionine to initiate H atom abstraction reactions. This remarkable chemistry involves the generation of the extremely reactive 5'-deoxyadenosyl radical, the same radical intermediate utilized in B12-dependent reactions. Although early speculation focused on the possibility of an organometallic intermediate in radical-SAM reactions, current evidence points to novel chemistry involving a site-differentiated [4Fe-4S] cluster. The focus of this forum article is on one member of the radical-SAM superfamily, pyruvate formate-lyase activating enzyme, and how physical methods, primarily EPR and ENDOR spectroscopies, are contributing to our understanding of its structure and mechanism. New ENDOR data supporting coordination of the methionine moiety of SAM to the unique site of the [4Fe-4S]2+/+ cluster are presented.

Binding Sites↗

PubMed2005

Substrate modulation of the properties and reactivity of the oxy-ferrous and hydroperoxo-ferric intermediates of cytochrome P450cam as shown by cryoreduction-EPR/ENDOR spectroscopy.

EPR/ENDOR studies have been carried out on oxyferrous cytochrome P450cam one-electron cryoreduced by gamma-irradiation at 77 K in the absence of substrate and in the presence of a variety of substrates including its native hydroxylation substrate, camphor (a), and the alternate substrates, 5-methylenyl-camphor (b), 5,5-difluorocamphor (c), norcamphor (d), and adamantanone (e); the equivalent experiments have been performed on the T252A mutant complexed with a and b. The present study shows that the properties and reactivity of the oxyheme and of both the primary and the annealed intermediates are modulated by a bound substrate. This includes alterations in the properties of the heme center itself (g tensor; (14)N, (1)H, hyperfine couplings). It also includes dramatic changes in reactivity: the presence of any substrate increases the lifetime of hydroperoxoferri-P450cam (2) no less than ca. 20-fold. Among the substrates, b stands out as having an exceptionally strong influence on the properties and reactivity of the P450cam intermediates, especially in the T252A mutant. The intermediate, 2(T252A)-b, does not lose H(2)O(2), as occurs with 2(T252A)-a, but decays with formation of the epoxide of b. Thus, these observations show that substrate can modulate the properties of both the monoxygenase active-oxygen intermediates and the proton-delivery network that encompasses them.

Adamantane↗

PubMed2005

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.

Acyl Carrier Protein↗