Biotin synthase catalyzes the insertion of a sulfur atom between the saturated C6 and C9 carbons of dethiobiotin. Catalysis requires AdoMet and flavodoxin and generates 5'-deoxyadenosine and methionine, suggesting that biotin synthase is an AdoMet-dependent radical enzyme. Biotin synthase (BioB) is aerobically purified as a dimer of 38.4 kDa monomers that contains 1-1.5 [2Fe-2S](2+) clusters per monomer and can be reconstituted with exogenous iron, sulfide, and reductants to contain up to two [4Fe-4S] clusters per monomer. The iron-sulfur clusters may play a dual role in biotin synthase: a reduced iron-sulfur cluster is probably involved in radical generation by mediating the reductive cleavage of AdoMet, while recent in vitro labeling studies suggest that an iron-sulfur cluster also serves as the immediate source of sulfur for the biotin thioether ring. Consistent with this dual role for iron-sulfur clusters in biotin synthase, we have found that the protein is stable, containing one [2Fe-2S](2+) cluster and one [4Fe-4S](2+) cluster per monomer. In the present study, we demonstrate that this mixed cluster state is essential for optimal activity. We follow changes in the Fe and S content and UV/visible and EPR spectra of the enzyme during a single turnover and conclude that during catalysis the [4Fe-4S](2+) cluster is preserved while the [2Fe-2S](2+) cluster is destroyed. We propose a mechanism for incorporation of sulfur into dethiobiotin in which a sulfur atom is oxidatively extracted from the [2Fe-2S](2+) cluster.

The biological activity of nitric oxide (NO) and NO-donors has been extensively investigated yet few studies have examined those of nitroxyl (HNO) species even though both exist in chemical equilibrium but oxidize thiols by different reaction mechanisms: S-nitrosation versus disulfide bond formation. Here, sodium trioxodinitrate (Na2N2O3; Angeli's salt; ANGS) was used as an HNO donor to investigate its effects on skeletal (RyR1) and cardiac (RyR2) ryanodine receptors. At steady-state concentrations of nanomoles/L, HNO induced a rapid Ca2+ release from sarcoplasmic reticulum (SR) vesicles then the reducing agent dithiothreitol (DTT) reversed the oxidation by HNO resulting in Ca2+ re-uptake by SR vesicles. With RyR1 channel proteins reconstituted in planar bilayers, HNO added to the cis-side increased the open probability (Po) from 0.056+/-0.026 to 0.270+/-0.102 (P<0.005, n=4) then DTT (3 mM) reduced Po to 0.096+/-0.040 (P<0.01, n=4). In parallel experiments, the time course of HNO production from ANGS was monitored by EPR and UV spectroscopy and compared with the rate of SR Ca2+ release indicating that picomolar concentrations of HNO triggered SR Ca2+ release. Controls showed that the hydroxyl radical scavenger, phenol did not alter ANGS-induced SR Ca2+ release, indicating that hydroxyl radical production from ANGS did not account for Ca2+ release from the SR. The findings indicate that HNO is a more potent activator of RyR1 than NO and that HNO activation of RyRs may contribute to NO's activation of RyRs and to the therapeutic effects of HNO-releasing prodrugs in heart failure.

Solid tumor has unique vascular architecture, excessive production of vascular mediators, and extravasation of macromolecules from blood vessels into the tumor tissue interstitium. These features comprise the phenomenon named the EPR effect of solid tumors, described in 1986. Our investigations on the EPR revealed that many mediators, such as bradykinin, NO, and prostaglandins, are involved in the EPR effect, which is now believed to be the most important element for cancer-selective drug delivery. However, tumors in vivo manifest great diversity, and some demonstrate a poor EPR effect, for example, because of impaired vascular flow involving thrombosis, with poor drug delivery and therapeutic failure. Another important element of this effect is that it operates in metastatic cancers. Because few drugs are currently effective against metastases, the EPR effect offers a great advantage in nanomedicine therapy. The EPR effect can also be augmented two to three times via nitroglycerin, ACE inhibitors, and angiotensin II-induced hypertension. The delivery of nanomedicines to tumors can thereby be enhanced. In traditional PDT, most PSs had low MW and little tumor-selective accumulation. Our hydroxypropylmetacrylamide-polymer-conjugated-PS, zinc protoporphyrin (apparent MW >50 kDa) showed tumor-selective accumulation, as revealed by fluorescent imaging of autochthonous cancers. After one i.v. injection of polymeric PS followed by two or three xenon light irradiation/treatments, most tumors regressed. Thus, nanoprobes with the EPR effect seem to have remarkable effects. Enhancing the EPR effect by using vascular modulators will aid innovations in PDT for greater tumor-targeted drug delivery.

Micelles prepared from polyethyleneglycol/phosphatidyl-ethanolamine conjugates (PEG-PE) with a size of 7-20 nm and zeta-potential of approximately -18 mV were administered i.v. to rabbits with experimental myocardial infarctions. Micelles demonstrated a prolonged circulation in the blood (half-life of 2 h) and accumulated in the infarction zone with efficiency more than 8-fold higher as compared to a non-damaged part of the heart muscle. Obtained results suggest that the enhanced permeability and retention (EPR) effect is the primary mechanism of accumulation of microparticles in the infarct areas, and that drug carriers such as PEG-PE micelles can be used for the delivery of therapeutic or diagnostic agents to an area of myocardial infarction.

The generation of reactive oxygen species in mitochondria acts as a redox signal in triggering cellular events such as apoptosis, proliferation, and senescence. Overproduction of superoxide (O2*-) and O2*--derived oxidants changes the redox status of the mitochondrial GSH pool. An electron transport protein, mitochondrial complex I, is the major host of reactive/regulatory protein thiols. An important response of protein thiols to oxidative stress is to reversibly form protein mixed disulfide via S-glutathiolation. Exposure of complex I to oxidized GSH, GSSG, resulted in specific S-glutathiolation at the 51 kDa and 75 kDa subunits (Beer et al. (2004) J. Biol. Chem. 279, 47939-47951). Here, to investigate the molecular mechanism of S-glutathiolation of complex I, we prepared isolated bovine complex I under nonreducing conditions and employed the techniques of mass spectrometry and EPR spin trapping for analysis. LC/MS/MS analysis of tryptic digests of the 51 kDa and 75 kDa polypeptides from glutathiolated complex I (GS-NQR) revealed that two specific cysteines (C206 and C187) of the 51 kDa subunit and one specific cysteine (C367) of the 75 kDa subunit were involved in redox modifications with GS binding. The electron transfer activity (ETA) of GS-NQR in catalyzing NADH oxidation by Q1 was significantly enhanced. However, O2*- generation activity (SGA) mediated by GS-NQR suffered a mild loss as measured by EPR spin trapping, suggesting the protective role of S-glutathiolation in the intact complex I. Exposure of NADH dehydrogenase (NDH), the flavin subcomplex of complex I, to GSSG resulted in specific S-glutathiolation on the 51 kDa subunit. Both ETA and SGA of S-glutathiolated NDH (GS-NDH) decreased in parallel as the dosage of GSSG increased. LC/MS/MS analysis of a tryptic digest of the 51 kDa subunit from GS-NDH revealed that C206, C187, and C425 were glutathiolated. C425 of the 51 kDa subunit is a ligand residue of the 4Fe-4S N3 center, suggesting that destruction of 4Fe-4S is the major mechanism involved in the inhibition of NDH. The result also implies that S-glutathiolation of the 75 kDa subunit may play a role in protecting the 4Fe-4S cluster of the 51 kDa subunit from redox modification when complex I is exposed to redox change in the GSH pool.

Thermally responsive elastin-like polypeptides (ELPs) are a promising class of recombinant biopolymers for the delivery of drugs and imaging agents to solid tumors via systemic or local administration. This article reviews four applications of ELPs to drug delivery, with each delivery mechanism designed to best exploit the relationship between the characteristic transition temperature (T(t)) of the ELP and body temperature (T(b)). First, when T(t)≫T(b), small hydrophobic drugs can be conjugated to the C-terminus of the ELP to impart the amphiphilicity needed to mediate the self-assembly of nanoparticles. These systemically delivered ELP-drug nanoparticles preferentially localize to the tumor site via the <em>EPR</em> effect, resulting in reduced toxicity and enhanced treatment efficacy. The remaining three approaches take direct advantage of the thermal responsiveness of ELPs. In the second strategy, where T(b)<T(t)<42°C, an ELP-drug conjugate can be injected in conjunction with external application of mild hyperthermia to the tumor to induce ELP coacervation and an increase in concentration within the tumor vasculature. The third approach utilizes hydrophilic-hydrophobic ELP block copolymers that have been designed to assemble into nanoparticles in response to hyperthermai due to the independent thermal transition of the hydrophobic block, thus resulting in multivalent ligand display of a ligand for spatially enhanced vascular targeting. In the final strategy, ELPs with T(t)<T(b) are conjugated with radiotherapeutics, injtect intioa tumor where they undergo coacervation to form an injectable drug depot for intratumoral delivery. These injectable coacervate ELP-radionuclide depots display a long residence in the tumor and result in inhibition of tumor growth.

In a previous communication [Hu et al., J. Am. Chem. Soc. 126, 10844 (2004)], an approach was demonstrated that improves the efficiency of the cross-effect polarization mechanism employed in high field dynamic nuclear polarization (DNP) experiments. Specifically, it was shown that tethering two TEMPO (2,2,6,6-tetramethylpiperidin-1-oxyl) radicals increases the electron-electron dipole coupling from approximately 1 MHz in solutions of monomeric TEMPO to approximately 25 MHz in a tethered biradical. The larger coupling resulted in an increase in the DNP enhancements by a factor of approximately 3-4, from 45-50 to approximately 165. Here, a second approach to improving the efficiency of the polarization process is described that involves approximately satisfying the matching condition |omega(2e)-omega(1e)|=omega(n), where omega(2e) and omega(1e) are two frequencies in the electron paramagnetic resonance (EPR) spectrum and omega(n) is the Larmor frequency of the nuclear spins being polarized. Specifically, in a mixture of TEMPO and trityl [tris (8-carboxy-2,2,6,6-tetramethyl(-d3)-benzo[1,2d:4,5-d']bis(1,3)dithiol-4-yl) methyl] radicals, the intensity maxima in the EPR spectra of these two species are approximately separated by the (1)H NMR frequency. In this case the frequency difference between the g(yy) value of TEMPO and the narrow pseudo-isotropic g-value of trityl is approximately 224 MHz and the (1)H Larmor frequency is 211 MHz. The optimal magnetic field for DNP using the mixtures was found to coincide with the trityl EPR resonance. At 90 K and 5 T, a mixture of 20 mM TEMPO and 20 mM trityl enhanced the (1)H polarization by a factor of approximately 160, an improvement over the enhancement of approximately 50 with 40 mM TEMPO. The reasons for the improvement are discussed and evidence is presented suggesting that DNP enhancement can be improved further by tethering TEMPO and trityl or two similar radicals.

A method was developed to determine the interspin distances of two or more nitroxide spin labels attached to specific sites in proteins. This method was applied to different conformations of spin-labeled insulins. The electron paramagnetic resonance (EPR) line broadening due to dipolar interaction is determined by fitting simulated EPR powder spectra to experimental data, measured at temperatures below 200 K to freeze the protein motion. The experimental spectra are composed of species with different relative nitroxide orientations and interspin distances because of the flexibility of the spin label side chain and the variety of conformational substates of proteins in frozen solution. Values for the average interspin distance and for the distance distribution width can be determined from the characteristics of the dipolar broadened line shape. The resulting interspin distances determined for crystallized insulins in the R6 and T6 structure agree nicely with structural data obtained by x-ray crystallography and by modeling of the spin-labeled samples. The EPR experiments reveal slight differences between crystal and frozen solution structures of the B-chain amino termini in the R6 and T6 states of hexameric insulins. The study of interspin distances between attached spin labels can be applied to obtain structural information on proteins under conditions where other methods like two-dimensional nuclear magnetic resonance spectroscopy or x-ray crystallography are not applicable.

The Galpha subunits of heterotrimeric G proteins (Galphabetagamma) mediate signal transduction via activation by receptors and subsequent interaction with downstream effectors. Crystal structures indicate that conformational changes in "switch" sequences of Galpha, controlled by the identity of the bound nucleotide (GDP and GTP), modulate binding affinities to the Gbetagamma subunits, receptor, and effector proteins. To investigate the solution structure and dynamics of Galphai1 through the G protein cycle, nitroxide side chains (R1) were introduced at sites in switch II and at a site in helix alpha4, a putative effector binding region. In the inactive Galphai1(GDP) state, the EPR spectra are compatible with conformational polymorphism in switch II. Upon complex formation with Gbetagamma, motions of R1 are highly constrained, reflecting direct contact interactions at the Galphai1-Gbeta interface; remarkably, the presence of R1 at the sites investigated does not substantially affect the binding affinity. Complex formation between the heterotrimer and activated rhodopsin leads to a dramatic change in R1 motion at residue 217 in the receptor-binding alpha2/beta4 loop and smaller allosteric changes at the Galphai1-Gbetagamma interface distant from the receptor binding surface. Upon addition of GTPgammaS, the activated Galphai1(GTP) subunit dissociates from the complex, and switch II is transformed to a unique conformation similar to that in crystal structures but with a flexible backbone. A previously unreported activation-dependent change in alpha4, distant from the interaction surface, supports a role for this helix in effector binding.

Bacterioferritins are members of a class of spherical shell-like iron storage proteins that catalyze the oxidation and hydrolysis of iron at specific sites inside the protein shell, resulting in formation of a mineral core of hydrated ferric oxide within the protein cavity. Electrode oximetry/pH stat was used to study iron oxidation and hydrolysis chemistry in E. coli bacterioferritin. Consistent with previous UV-visible absorbance measurements, three distinct kinetic phases were detected, and the stoichiometric equations corresponding to each have been determined. The rapid phase 1 reaction corresponds to pairwise binding of 2 Fe(2+) ions at a dinuclear site, called the ferroxidase site, located within each of the 24 subunits, viz., 2Fe(2+) + P(Z) ->> [Fe(2)-P](Z) + 4H(+), where P(Z) is the apoprotein of net charge Z and [Fe(2)-P](Z) represents a diferrous ferroxidase complex. The slower phase 2 reaction corresponds to the oxidation of this complex by molecular oxygen according to the net equation: [Fe(2)-P](Z) + (1)/(2)O(2) ->> [Fe(2)O-P](Z) where [Fe(2)O-P](Z) represents an oxidized diferric ferroxidase complex, probably a mu-oxo-bridged species as suggested by UV-visible and EPR spectrometric titration data. The third phase corresponds to mineral core formation according to the net reaction: 4Fe(2+) + O(2) + 6H(2)O ->> 4FeO(OH)((core)) + 8H(+). Iron oxidation is inhibited by the presence of Zn(2+) ions. The patterns of phase 2 and phase 3 inhibition are different, though inhibition of both phases is complete at 48 Zn(2+)per 24mer, i.e., 2 Zn(2+) per ferroxidase center.

Biological O(2) activation often occurs after binding to a reduced metal [e.g., M(II)] in an enzyme active site. Subsequent M(II)-to-O(2) electron transfer results in a reactive M(III)-superoxo species. For the extradiol aromatic ring-cleaving dioxygenases, we have proposed a different model where an electron is transferred from substrate to O(2) via the M(II) center to which they are both bound, thereby obviating the need for an integral change in metal redox state. This model is tested by using homoprotocatechuate 2,3-dioxygenases from Brevibacterium fuscum (Fe-HPCD) and Arthrobacter globiformis (Mn-MndD) that share high sequence identity and very similar structures. Despite these similarities, Fe-HPCD binds Fe(II) whereas Mn-MndD incorporates Mn(II). Methods are described to incorporate the nonphysiological metal into each enzyme (Mn-HPCD and Fe-MndD). The x-ray crystal structure of Mn-HPCD at 1.7 A is found to be indistinguishable from that of Fe-HPCD, while EPR studies show that the Mn(II) sites of Mn-MndD and Mn-HPCD, and the Fe(II) sites of the NO complexes of Fe-HPCD and Fe-MndD, are very similar. The uniform metal site structures of these enzymes suggest that extradiol dioxygenases cannot differentially compensate for the 0.7-V gap in the redox potentials of free iron and manganese. Nonetheless, all four enzymes exhibit nearly the same K(M) and V(max) values. These enzymes constitute an unusual pair of metallo-oxygenases that remain fully active after a metal swap, implicating a different way by which metals are used to promote oxygen activation without an integral change in metal redox state.

[Fe(IV)(O)(TMG(3)tren)](2+) (1; TMG(3)tren = 1,1,1-tris{2-[N(2)-(1,1,3,3-tetramethylguanidino)]ethyl}amine) is a unique example of an isolable synthetic S = 2 oxoiron(IV) complex, which serves as a model for the high-valent oxoiron(IV) intermediates observed in nonheme iron enzymes. Congruent with DFT calculations predicting a more reactive S = 2 oxoiron(IV) center, 1 has a lifetime significantly shorter than those of related S = 1 oxoiron(IV) complexes. The self-decay of 1 exhibits strictly first-order kinetic behavior and is unaffected by solvent deuteration, suggesting an intramolecular process. This hypothesis was supported by ESI-MS analysis of the iron products and a significant retardation of self-decay upon use of a perdeuteromethyl TMG(3)tren isotopomer, d(36)-1 (KIE = 24 at 25 degrees C). The greatly enhanced thermal stability of d(36)-1 allowed growth of diffraction quality crystals for which a high-resolution crystal structure was obtained. This structure showed an Fe horizontal lineO unit (r = 1.661(2) A) in the intended trigonal bipyramidal geometry enforced by the sterically bulky tetramethylguanidinyl donors of the tetradentate tripodal TMG(3)tren ligand. The close proximity of the methyl substituents to the oxoiron unit yielded three symmetrically oriented short C-D...O nonbonded contacts (2.38-2.49 A), an arrangement that facilitated self-decay by rate-determining intramolecular hydrogen atom abstraction and subsequent formation of a ligand-hydroxylated iron(III) product. EPR and Mossbauer quantification of the various iron products, referenced against those obtained from reaction of 1 with 1,4-cyclohexadiene, allowed formulation of a detailed mechanism for the self-decay process. The solution of this first crystal structure of a high-spin (S = 2) oxoiron(IV) center represents a fundamental step on the path toward a full understanding of these pivotal biological intermediates.

OBJECTIVE

Tumor hypoxia has long been known to produce resistance to radiation. In this study, electron paramagnetic resonance (EPR) oxygen imaging was investigated for its power to predict the success of tumor control according to tumor oxygenation level and radiation dose.

METHODS

A total of 34 EPR oxygen images were obtained from the legs of C3H mice bearing 0.5-cm(3) FSa fibrosarcomas under both normal (air breathing) and clamped tumor conditions. Under the same conditions as those during which the images were obtained, the tumors were irradiated to a variety of doses near the FSa dose at which 50% of tumors were cured. Tumor tissue was distinguished from normal tissue using co-registration of the EPR oxygen images with spin-echo magnetic resonance imaging of the tumor and/or stereotactic localization. The tumor voxel statistics in the EPR oxygen image included the mean and median partial pressure of oxygen and the fraction of tumor voxels below the specified partial pressure of oxygen values of 3, 6, and 10 mm Hg. Bivariate logistic regression analysis using the radiation dose and each of the EPR oxygen image statistics to determine which best separated treatment failure from success.

RESULTS

The measurements of the dose at which 50% of tumors were cured were similar to those found in published data for this syngeneic tumor. Bivariate analysis of 34 tumors demonstrated that tumor cure correlated with dose (p = 0.004) and with a <10 mm Hg hypoxic fraction (p = 0.023).

CONCLUSIONS

Our results have shown that, together, radiation dose and EPR image hypoxic fraction separate the population of FSa fibrosarcomas that are cured from those that fail, thus predicting curability.

Glt(Ph) is a Pyrococcus horikoshii homotrimeric Na(+)-coupled aspartate transporter that belongs to the glutamate transporter family. Each protomer consists of a trimerization domain involved in subunit interaction and a transporting domain with the substrate-binding site. Here, we have studied the conformational changes underlying transport by Glt(Ph) using EPR spectroscopy. The trimerization domains form a rigid scaffold, whereas the transporting domains sample multiple conformations, consistent with large-scale movements during the transport cycle. Binding of substrates changed the occupancies of the different conformational states, but the domains remained heterogeneous. The membrane environment favored conformations different from those observed in detergent micelles, but the transporting domain remained structurally heterogeneous in both environments. We conclude that the transporting domains sample multiple conformational states with substantial occupancy regardless of the presence of substrate and coupling ions, consistent with equilibrium constants close to unity between the observed transporter conformations.

Talin is a key protein involved in linking integrins to the actin cytoskeleton. The long flexible talin rod domain contains a number of binding sites for vinculin, a cytoskeletal protein important in stabilizing integrin-mediated cell-matrix junctions. Here we report the solution structure of a talin rod polypeptide (residues 1843-1973) which contains a single vinculin binding site (VBS; residues 1944-1969). Like other talin rod polypeptides, it consists of a helical bundle, in this case a four-helix bundle with a right-handed topology. The residues in the VBS important for vinculin binding were identified by studying the binding of a series of VBS-related peptides to the vinculin Vd1 domain. The key binding determinants are buried in the interior of the helical bundle, suggesting that a substantial structural change in the talin polypeptide is required for vinculin binding. Direct evidence for this was obtained by NMR and EPR spectroscopy. [1H,15N]-HSQC spectra of the talin fragment indicate that vinculin binding caused approximately two-thirds of the protein to adopt a flexible random coil. For EPR spectroscopy, nitroxide spin labels were attached to the talin polypeptide via appropriately located cysteine residues. Measurements of inter-nitroxide distances in doubly spin-labeled protein showed clearly that the helical bundle is disrupted and the mobility of the helices, except for the VBS helix, is markedly increased. Binding of vinculin to talin is thus a clear example of the unusual phenomenon of protein unfolding being required for protein/protein interaction.

The fate of vanadate (+5 oxidation state of vanadium) taken up by the red cell was studied using EPR spectroscopy. The appearance of an EPR signal indicated that most of the cytoplasmic vanadate is reduced to the +4 oxidation state with axial symmetry characteristic of vanadyl ions. The signal at 23 degrees C was characteristic of an immobilized system indicating that the vanadyl ions in the cytoplasm are associated with a large molecule. [48V]Vanadium eluted with hemoglobin when the lysate from Na3[48V[O4-treated red cells was passed through a Sephadex G-100 column and rabbit anti-human hemoglobin serum caused a hemoglobin-specific precipitation of 48V when added to the red cell lysate. Both results indicate that hemoglobin is the protein which binds cytoplasmic vanadyl ions. However, neither sodium vanadate nor vanadyl sulfate bound to purified hemoglobin in vitro. Finally, transient kinetics of vanadyl sulfate interaction with the sodium-and potassium-stimulated adenosine triphosphatase showed that the +4 oxidation state of vanadium is less effective than the +5 oxidation state in inhibiting this enzyme. These results indicate that oxidation-reduction reactions in the cytoplasm are capable of relieving vanadate inhibition of cation transport.

The SF-Abs, RFQ-EPR, and RFQ-Möss data on the R2 reconstitution reaction are all consistent with the mechanism of Scheme I, in which the intermediate X is the immediate precursor to the product cofactor, and illustrate how the continuous SF approach and the discontinuous RFQ methods can be complementary. Given the inherent differences in the methods, it should not be taken for granted that data from the two will be consistent. A number of problems can be associated with the RFQ approach. For example, isopentane could conceivably interfere with or alter the chemistry to be studied. A second potential problem involves temperature-dependent equilibria among different intermediate species. This problem has been encountered by Dooley et al. with the 6-hydroxydopa-requiring protein, plasma amine oxidase and was previously observed with the adenosylcobalamin-dependent ribonucleotide reductase by Blakley and co-workers. This potential complication should be considered when discrepancies arise between SF and RFQ data and in low temperature structural studies of reactive intermediates in general. Each of the three methods employed can yield time-resolved quantitation of reaction components. In this regard, SF-Abs has the disadvantage of poor resolution, such that quantitation of individual components most often requires sophisticated mathematical analysis. Obvious advantages to the RFQ-Möss method are the presence of an internal standard (the known amount of 57Fe being proportional to the total absorption area) and the spectroscopic activity of all reaction components which contain iron. In our hands, quantitation by RFQ-EPR was most problematic and least reproducible. This irreproducibility most likely relates to heterogeneity among samples in terms of volume and density. As discussed in detail by Ballou and Palmer, the packing factor, which relates to the fraction of a sample made up by the reaction solution (the remainder being frozen isopentane), is dependent on the investigator. Given this caveat, it is not surprising that the RFQ-EPR data had the greatest uncertainty in our hands. Placing a chemically unreactive, EPR active standard in each reaction mixture could help alleviate this problem. Time-resolved Möss methods can be extremely powerful if excellent, nonoverlapping reference spectra of starting materials, products, and intermediates are available. All of the iron centers can be examined simultaneously. The problems associated with Möss arise from its extreme insensitivity. It takes millimolar solutions of proteins and several days for data collection of each time point.(ABSTRACT TRUNCATED AT 400 WORDS)

Five mutations of rhodopsin have been produced, each of which contains a unique cysteine residue at positions 62, 65, 140, 240, or 316 in the cytoplasmic domain. The single reactive cysteines were derivatized with a sulfhydryl-specific nitroxide spin-label, and the electron paramagnetic resonance (EPR) spectra were analyzed in both lauryl maltoside and digitonin in the dark and after photobleaching. The collision rate of the attached nitroxides with polar and nonpolar paramagnetic agents indicated that they were all exposed to the aqueous environment. Photobleaching of the mutants in digitonin, which arrests the protein at the meta I intermediate, produced little change in mobility of the attached nitroxide. On the other hand, photobleaching in lauryl maltoside produced the meta II intermediate and significant changes in the EPR spectra of the nitroxides attached to positions 140 and 316. These data directly reveal a light-induced conformational change in the cytoplasmic loops that accompanies meta II formation.

The role of the high potential [3Fe-4S]1+,0 cluster of [NiFe] hydrogenase from Desulfovibrio species located halfway between the proximal and distal low potential [4Fe-4S]2+,1+ clusters has been investigated by using site-directed mutagenesis. Proline 238 of Desulfovibrio fructosovorans [NiFe] hydrogenase, which occupies the position of a potential ligand of the lacking fourth Fe-site of the [3Fe-4S] cluster, was replaced by a cysteine residue. The properties of the mutant enzyme were investigated in terms of enzymatic activity, EPR, and redox properties of the iron-sulfur centers and crystallographic structure. We have shown on the basis of both spectroscopic and x-ray crystallographic studies that the [3Fe-4S] cluster of D. fructosovorans hydrogenase was converted into a [4Fe-4S] center in the P238 mutant. The [3Fe-4S] to [4Fe-4S] cluster conversion resulted in a lowering of approximately 300 mV of the midpoint potential of the modified cluster, whereas no significant alteration of the spectroscopic and redox properties of the two native [4Fe-4S] clusters and the NiFe center occurred. The significant decrease of the midpoint potential of the intermediate Fe-S cluster had only a slight effect on the catalytic activity of the P238C mutant as compared with the wild-type enzyme. The implications of the results for the role of the high-potential [3Fe-4S] cluster in the intramolecular electron transfer pathway are discussed.

Protocatechuate 4,5-dioxygenase from Pseudomonas testosteroni has been purified to homogeneity and crystallized. The iron containing, extradiol dioxygenase is shown to be composed of two subunit types (alpha, Mr = 17,700 and beta, Mr = 33,800) in a 1:1 ratio; such a composition has not been observed for other extradiol dioxygenases. The 4.2 K Mössbauer spectrum of native protocatechuate 4,5-dioxygenase prepared from cells grown in 57Fe-enriched media consists of a doublet with quadrupole splitting, delta EQ = 2.22 mm/s, and isomer shift delta Fe = 1.28 mm/s, demonstrating a high spin Fe2+ site. These parameters, and the temperature dependence of delta EQ, are unique among enzymes but are strikingly similar to those reported for the reaction center of the photosynthetic bacterium Rhodopseudomonas sphaeroides R-26, suggesting very similar ligand environments. The Fe2+ of protocatechuate 4,5-dioxygenase can be oxidized, for instance by H2O2, to yield high spin Fe3+ with EPR g values around g = 6 (and g = 4.3). In the oxidized state, protocatechuate 4,5-dioxygenase is inactive; the iron, however, can be rereduced by ascorbate to yield active enzyme. Our data suggest that protocatechuate binds to Fe2+; the spectra indicate that the ligand binding is heterogenous. The Mössbauer spectra observed here are fundamentally different from those reported earlier (Zabinski, R., Münck, E., Champion, P., and Wood, J. M. (1972) Biochemistry 11, 3212-3219). The spectra of the earlier (reconstituted) preparations, which had substantially lower specific activities, probably reflect adventitiously bound Fe3+. We discuss here how adventitiously bound iron can be identified and removed. The Fe2+ which is present in native protocatechuate 4,5-dioxygenase and its complexes with substrates and inhibitors reacts quantitatively with nitric oxide to produce a species with electronic spin S = 3/2. The EPR and Mössbauer spectra of these complexes compare favorably with EDTA . Fe(II) . NO. We have studied the latter complex extensively and have analyzed the Mössbauer spectra with an S = 3/2 spin Hamiltonian. EPR spectra show that protocatechuate 4,5-dioxygenase-NO complexes with substrates or inhibitors are heterogeneous and consist of several well defined subspecies. The data show that NO, and presumably also O2, has access to the active site Fe2+ in the enzyme-substrate complex. The use of EPR-detectable NO complexes as a rapid and sensitive tool for the study of the EPR silent active site iron of extradiol dioxygenases is discussed.

Hydrogenases are enzymes catalyzing the reversible heterolytic splitting of molecular hydrogen. Despite extensive investigations of this class of enzymes its catalytic mechanism is not yet well understood. In this paper spectroscopic investigations of the active site of [FeFe] hydrogenase are presented. The so-called H-cluster consists of a bi-nuclear catalytically active subcluster connected to a [4Fe4S] ferredoxin-like unit via a Cys-thiol bridge. An important feature of the H-cluster is that both irons in the bi-nuclear subcluster are coordinated by CN and CO ligands. The bi-nuclear site also carries a dithiol bridge, whose central atom has not yet been identified. Nitrogen and oxygen are the most probable candidates from a mechanistic point of view. Here we present a study of the (14)N nuclear quadrupole and hyperfine interactions of the active oxidized state of the H-cluster using advanced EPR methods. In total three (14)N nuclei with quadrupole couplings of 0.95 MHz, 0.35 MHz and 1.23 MHz were detected using hyperfine sublevel correlation spectroscopy (HYSCORE). The assignment of the signals is based on their (14)N quadrupole couplings in combination with DFT calculations. One signal is assigned to the CN ligand of the distal iron, one to a Lys side chain nitrogen and one to the putative nitrogen of the dithiol bridge. Hence, these results provide the first experimental evidence for a di-(thiomethyl)amine ligand (-S-CH(2)-NH-CH(2)-S-) in the bi-nuclear subcluster. This finding is important for understanding the mechanism of [FeFe] hydrogenases, since the nitrogen is likely to act as an internal base facilitating the heterolytic splitting/formation of H(2).

Yah1p, an [Fe 2S 2]-containing ferredoxin located in the matrix of Saccharomyces cerevisiae mitochondria, functions in the synthesis of Fe/S clusters and heme a prosthetic groups. EPR, Mossbauer spectroscopy, and electron microscopy were used to characterize the Fe that accumulates in Yah1p-depleted isolated intact mitochondria. Gal- YAH1 cells were grown in standard rich media (YPD and YPGal) under O 2 or argon atmospheres. Mitochondria were isolated anaerobically, then prepared in the as-isolated redox state, the dithionite-treated state, and the O 2-treated state. The absence of strong EPR signals from Fe/S clusters when Yah1p was depleted confirms that Yah1p is required in Fe/S cluster assembly. Yah1p-depleted mitochondria, grown with O 2 bubbling through the media, accumulated excess Fe (up to 10 mM) that was present as 2-4 nm diameter ferric nanoparticles, similar to those observed in mitochondria from yfh1Delta cells. These particles yielded a broad isotropic EPR signal centered around g = 2, characteristic of superparamagnetic relaxation. Treatment with dithionite caused Fe (3+) ions of the nanoparticles to become reduced and largely exported from the mitochondria. Fe did not accumulate in mitochondria isolated from cells grown under Ar; a significant portion of the Fe in these organelles was in the high-spin Fe (2+) state. This suggests that the O 2 used during growth of Gal- YAH1 cells is responsible, either directly or indirectly, for Fe accumulation and for oxidizing Fe (2+) ->> Fe (3+) prior to aggregation. Models are proposed in which the accumulation of ferric nanoparticles is caused either by the absence of a ligand that prevents such precipitation in wild-type mitochondria or by a more oxidizing environment within the mitochondria of Yah1p-depleted cells exposed to O 2. The efficacy of reducing accumulated Fe along with chelating it should be considered as a strategy for its removal in diseases involving such accumulations.

The region 35-43 of human alpha-Synuclein bound to small unilamellar lipid vesicles and to sodium dodecyl sulfate micelles has been investigated by site-directed spin labeling and electron paramagnetic resonance spectroscopy. The distance distributions obtained from spectral fitting have been analyzed on the basis of the allowed rotamers of the spin-label side-chain. Very similar results have been obtained in the two environments: an unbroken helical structure of the investigated region can be ruled out. The distance distributions are rather compatible with the presence of conformational disorder, in agreement with previous findings for micelle-bound alpha-Synuclein. The propensity for helix breaking is confirmed by molecular dynamics simulations.

Electron paramagnetic resonance (EPR) spectroscopy has often played a crucial role in characterizing the various cofactors and processes of photosynthesis, and photosystem II and its oxygen evolving chemistry is no exception. Until recently, the application of EPR spectroscopy to the characterization of the oxygen evolving complex (OEC) has been limited to the S2-state of the Kok cycle. However, in the past few years, continuous wave-EPR signals have been obtained for both the S0- and S1-state as well as for the S2 (radical)(Z)-state of a number of inhibited systems. Furthermore, the pulsed EPR technique of electron spin echo electron nuclear double resonance spectroscopy has been used to directly probe the 55Mn nuclei of the manganese cluster. In this review, we discuss how the EPR data obtained from each of these states of the OEC Kok cycle are being used to provide insight into the physical and electronic structure of the manganese cluster and its interaction with the key tyrosine, Y(Z).