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The studies have shown that in both native and reconstituted [4Fe-4S] FNR, the reaction environment and the presence of [Fe.sup.2} and [Fe.sup.3} chelators have influenced the cluster conversion reactions. The sensitivity of FNR cluster conversion kinetics to chelators has disclosed mechanism by which the cell can fine-tune its response to the onset of aerobic conditions.

The site directed nitroxide spin labeling (SDSL) of cysteine residues were used to search for conformational changes at the cytosolic entrance to the proton channels of the heme-copper quinol oxidase (QO), cytochrome b[o.sub.3], Escherichia coli. The kinetic studies of mutants demonstrate that the redox gating of protons occurs deep within the D channel close to the heme-copper site implies that no motion is transmitted to the ends of the helices.

Results show that the water-soluble domain of the nitric oxide reductase subunit C exhibits a alpha-helical structure and contains a partially solvent-exposed heme. The heme iron is coordinated by histidine and methionine ligands in both. Data suggest that the soluble domain of subunit C interacts with the catalytic subunit B.

A magneto-optical study is undertaken of the mixed-valence single-molecule magnet [MnIV (sub 4)MnIII (sub 8) O12 L16] in which the ligands, L, are acetate (Mn12Ac) or the long-chain carboxylic acid, C14 H-29COOH(Mn12C15) that confers better solubility in organic solvents. The optical polarization and the selection rules for field magnetic circular dichroism (MCD) select a subset of molecular orientations with respect to the direction of field.

The crystal structure of the periplasmic penta-heme cytochrome c nitrite reductase of Escherichia coli shows that four hemes of the enzyme exhibit bis-histidine axial heme-iron ligation with the catalytic heme-iron showing a distal lysine ligand and a proximal oxygen ligand. The spectroscopic signals and redox potentials of individual hemes are discussed.

Research illustrates the interaction of carbon monoxide with the dinuclear center of reduced bacterrial nitric oxide reductase. Results reveal that the ligand binds to iron(II) heme b(sub)3 forming a stable complex, which suggests differences in the organization of this active site from other heme-copper oxidase superfamily.

The blue copper protein azurin was studied using optically detected electron paramagnetic resonance spectroscopy.

The action of bacterial nitric oxide reductase (NOR) on nitric oxide and nitrous oxide plays an integral role in the completion of the two-electron process undergone by these compounds. NOR is considered a diverged member of the heme-copper oxidase group since it exhibits a distinct electronic absorption spectrum. Experimental data derived from a Pavacocus denitrificans strain suggest that the oxygen reducing activity manifested by heme-copper oxidases is significantly attributed to the substitution of copper for non-heme iron in the dinuclear center.

The spectroscopic data indicate that paracoccus denitrificans periplasmic nitrate reductase exhibit distinct molybdenum (Mo) oxidation states comprised of Mo(VI), (V), and (IV) and possesses a Mo-cysteine ligand. A model is presented for the enzyme catalysis and Mo coordination during enzyme turnover.

A study was conducted on the fully oxidized nitric oxide reductase (NOR) from Pseudomonas stutzeri. The findings indicate a spin-spin interaction between the heme and another paramagnet as well as an unusual near-infrared MCD. Moreover, the results suggest that NOR is structurally related to heme-copper oxidases and that it has a heme/non-heme iron spin-coupled pair at the active site. Thus, the nature of the metal ion paired with the heme in the active site may be essential in determining the specificity of these enzymes.

Halide metathesis with silver nitrite can be used for either stepwise or complete substitution of all three chloride ligands in the iron(III) compound (NEt4)(HB(3,5-Me2pz)3FeCl) to produce the complexes (NEt4)(HB(3,5-Me2pz)3Fe(ONO)xCl3-x), x = 1 - 3, and (NEt4)(HB(3,5-Me2pz)3Fe(eta2-ONO)Cl). The above compounds were characterized by elemental analyses, electrochemistry and spectroscopy. The crystal structures of the ferric complexes had monodentate, O-bound nitrito ligands.

The properties of the cytochromes cdsub1 from Pseudomonas stutzeri and Thiosphaera pantotropha have been compared, using MCD and EPR techniques. The results are consistent with previous indications that enzymes from different sources do not have identical heme iron ligands in the oxidized state, indicating there there are several classes of cytochrome cdsub1.

Electronic absorption, MCD and EPR spectroscopy have been used to study the reaction of nitric oxide (NO) with fast cytochrome bo from Escherichia coli. Two distinct species were formed and it was revealed that broad EPR signals are lost. Heme o stays in the high-spin FeIII state during titration, and the results are consistent with binding of two NO molecules at CuBII.

The reactive 3Fe cluster in the 7Fe ferredoxin from Desulfovibrio africanus, incorporating Fe and other metals to to form cubanes (M3Fe-4S) has a non-cystein ligation to the added metal (M). Connectivities among interconversion with different M, the influence of pH and the reactivities of certain cluster oxidation levels have been studied using electrochemical and spectroscopic methods.

The binding of azide to the fast cytochrome bo of Escherichia coli was analyzed by electronic absorption spectroscopy. Room temperature changes produced insignificant alterations in the binding of azide to the subunit I or CuB of cytochrome bo which is composed of three metal centers, two heme proteins and one copper(II) ions. Furthermore, the immediate ligands of heme o exhibited small energy shifts during the binding of the azide to the CuB subunit of quinol oxidase.

Bis-methionine coordinated cytochrome b562 proteins with or without covalently attached heme were generated and characterized by multiple circular dichroism and electron paramagnetic resonance spectroscopies, and protein electrochemistry. In the oxidized state, the single variant (H102M) was high-spin and was coordinated by a methionine ligand. Likewise, the double variant (R98C/H102M) was bis-methionine coordinated in the ferrous state. In the oxidized state, the protein transformed from a slow-spin to a high-spin species but did not exhibit the methionine coordination state.

Characterization of the novel 'hyper-reduced' form of protein-bound (3Fe-4S) clusters showed that its formation is reversible. It was also determined that in the weakly acidic/neutral region of pH, the cluster consumes a total of three protons for the overall three-proton reduction of (3Fe-4S)(super 1+). In the two-electron reduction of (3Fe-4S)(super 0) in a range of proteins, the product is surprisingly inert each time and the pH-dependent reduction potential is in the region of -700 mV vs SHE at pH 7.

The variant form Trp51Ala of yeast cytochrome c peroxidase has three active site states, and conversion from one form to another is dependent on ionic strength, specific anions and temperature. The electronic and MCD spectra exhibited by LS1, the acidic form, indicates a low-spin, bis-histidine coordination for the iron in the heme. The neutral HS form indicates a high-spin heme Fe(III), while the LS2 form indicates a low-spin Fe(III) derivative. A model of the active site of the enzyme is proposed, based on the nuclear magnetic resonance spectroscopy and electron absorption studies.

A study was conducted on the tetraheme flavocytochrome c from Shewanella putrefaciens NCIMB400 with emphasis on the N-terminal cytochrome domain using electron paramagnetic resonance and magnetic circular dichroism. Stopped-flow measurements were also employed to investigate the presence of paired hemes. Results showed that the four hemes observed were low spin and had a nitrogeneous sixth ligand. Stopped-flow data further revealed that fumarate-dependent heme oxidation was biphasic.

The redox-coupled binding of a thiolate ligand to an Fe-S protein cluster has been characterized by voltammetry. The protein ligand of Ferrodoxin III which contains seven cysteines capable of ligating both (4Fe-4S) and (3Fe-4S) clusters, utilized for characterization has been isolated from the Desulfovibrio africanus strain. The kinetics and equilibrium constants of the ligation has also been measured. Findings show no definite interaction betweeen either of the protein clusters and thiolate. However, voltammetric characterization demonstrated the time-separated coupling of electron transfer to conformational change.

Temperature dependence of the magnetic circular dichroism (MCD) intensity is seen for the nitrite-treated form of cytochrome [cd.sub.1] nitrate reductase. The radical species is shown to be either a protein-bound radical generated by a nitrite ion reacting with a heme group resulting in a one-electron oxidation of an amino acid residue or a heme [d.sub.1] [Fe.sup.III]NO complex.

The analysis results for Streptomyces coelicolor WhiD purified anaerobically from Escherichia coli as a soluble protein containing a single [[4Fe-4S].sup.2+] cluster ligated by four cysteines are presented. A negligible activity exhibited by apo-WhiD in an insulin-based disulfide reductase assay demonstrated that it does not function as a general protein disulfide reductase.

The synthesis experiments have shown that while no Re-Zn alloy or solid solution is formed, a novel [Re.sub.3]Zn[N.sub.x] ordered solid solution is formed with nitrogen occupying Re-coordinated cages. The studies have shown that unlike pure [Re.sub.3]Zn, the novel hexagonal [Re.sub.3]Zn[N.sub.x] structure is stabilized by nitrogen bond formation with rhenium.

Synthesis experiments at 10 GPa and 1500 K followed by annealing at 770 K using Ge and Sn starting materials and ex-situ analysis have documented the recovery of a [Ge.sub.0.9][Sn.sub.0.1] solid solution.

Catenanes and rotaxanes are prepared by using the hard trivalent transition metal ion cobalt(III) as a template. The removal of the metal ion for both catenanes and rotaxanes via reduction under acidic conditions to the more labile Co(II) has given neutral interlocked molecules with well-defined co-conformations stabilized by intercomponent hydrogen bonding.

An algorithm is designed to search coiled-coil specificity space, along with a few modest multicomponent structures to show the ability to build peptide-based nanostructures from the bottom up. This concept can be expanded to longer coiled coils and larger number of domains and the extensions have provided the extra versatility and stability required for achieving programmed multicomponent self-assembling systems.

The rise to similar fast-relaxing broad features in the dual-mode X-band EPR spectra is caused by spin-coupled high-spin Fe(lll) heme O3 and CuB(ll) ion. The MCD data, independent of the EPR study shows the spin-coupling with the active site.

Research has been conducted on dodecametallic Cr(III) cluster with the ground spin state characterized by the spin Hamiltonian parameters. The authors describe the magnetic characterization of this cluster carried out via the use of EPR spectroscopy and micro-SQUID magnetometry.

Rotaxanes can be prepared from a class of cyclic peptides and nonpeptic threads. Cyclo(L-ProGly)4 and cyclo(L-ProGly)5 are prepared from cyclic peptides by a solid-phase backbone amide-linking approach, by removing the macrocycles from a rotaxane-forming reaction mixture through simple aqueous extraction.

'Single-molecule magnets' (SMMs) represent the ultimate limit of miniaturization of magnetic data storage media. An optical method is demonstrated for determining the single-ion ZFS within clusters and the relative orientations of the local and cluster magnetic axes are as important as the sign of the single-ion ZFS in determining whether cluster would be a SMM .

The perpendicular- and parallel-mode X-band EPR spectra of fluoride bo3 was simulated to determine the interaction between high-spin Fe(III) heme and copper(II) ion in the active site of heme-copper oxidases. Results showed a weak coupling between the two metal ions for a heme with a fluoride ligand and for fast, formate and azide derivatives of bo3. This indicates that the intervening exogenous ligands have a small influence in mediating the exchange interaction between the two ions.

High pressure can induce profound changes in solids. A significant barrier to new alloys and ceramics, however, is that targeted starting materials may not react with each other, even with the help of pressure. We. use nitrogen, in a new capacity, to incorporate two otherwise unreactive elements, Re and Zn, in the same structure when pressure alone does not suffice, without nitrogen altering the resulting backbone structure. Synthesis experiments up to 20 GPa and 1800 K show that while no Re-Zn alloy or solid solution is formed, a novel Re3ZnNx ordered solid solution is formed, at 20 GPa, with nitrogen occupying Re-coordinated cages. We put forth that unlike pure Re3Zn, our novel hexagonal Re3ZnNx structure is stabilized by nitrogen bond formation with rhenium. Pressure lifts the pronounced ambient Zn anisotropy, making it more compatible with Re and likely facilitating incorporation of the structure-stabilizing nitrogen anion. This methodology and result denote further options for removing impasses to material preparation, thus opening new avenues for synthesis. These can also be pursued with other ions including carbon, hydrogen, and oxygen, in addition to nitrogen. [ABSTRACT FROM AUTHOR]

The article discusses the designed α-helical tectons for constructing multicomponent synthetic biological systems. As stated, the method applies rules relating sequence and stability in dimeric coiled coils to produce peptidic tectons for use in self-assembly and synthetic biology. The method uses a reduced alphabet and considers only the g, a, d and e positions of the heptad repeat.

In Escherichia coli, the switch between aerobic and anaerobic metabolism is primarily controlled by the fumarate and nitrate reduction transcriptional regulator FNR. In the absence of O2, FNR binds a [4Fe-4S]2+ cluster, generating a transcriptionally active dimeric form. Exposure to O2 results in the conversion of the cluster to a [2Fe-2S]2+ form, leading to dissociation of the protein into transcriptionally inactive monomers. The [4Fe-4S]2+ to [2Fe-2S]2+ cluster conversion proceeds in two steps. Step 1 involves the one-electron oxidation of the cluster, resulting in the release of Fe2+ generating a [3Fe-4S]1+ cluster intermediate, and a superoxide ion. In step 2, the cluster intermediate spontaneously rearranges to form the [2Fe-2S]2+ cluster, with the release of a Fe3+ ion and two sulfide ions. Here, we demonstrate that, in both native and reconstituted [4Fe-4S] FNR, the reaction environment and, in particular, the presence of Fe2+ and/or Fe3+ chelators can influence significantly the cluster conversion reaction. We demonstrate that while the rate of step 1 is largely insensitive to chelators, that of step 2 is significantly enhanced by both Fe2+ and Fe3+ chelators. We show that, for reactions in Fe3+-coordinating phosphate buffer, step 2 is enhanced to the extent that step 1 becomes the rate determining step and the [3Fe-4S]1+ intermediate is no longer detectable. Furthermore, Fe3+ released during this step is susceptible to reduction in the presence of Fe2+ chelators. This work, which may have significance for the in vivo FNR cluster conversion reaction in the cell cytoplasm, provides an explanation for apparently contradictory results reported from different laboratories. [ABSTRACT FROM AUTHOR]

Examines the optical detection of spin polarization in single-molecule magnets. Evaporation of solvents to provide samples for variable-temperature and field magnetic circular dichroism (MCD); Adsorption of spectra in isotropic light; Measurement on MCD magnetization curves.

We report a simple reductive amination protocol to ligate two peptides, while simultaneously installing a β-turn mimic at the ligation junction. This strategy uses commercially available materials, mild chemical conditions, and a chemoselective ligation reaction of unprotected peptide substrates accessed through standard solid phase methods. This system was implemented in a designed β-hairpin system, and biophysical analysis demonstrates effective mimicry of the β-turn.

Improving our understanding of biological motors, both to fully comprehend their activities in vital processes, and to exploit their impressive abilities for use in bionanotechnology, is highly desirable. One means of understanding these systems is through the production of synthetic molecular motors. We demonstrate the use of orthogonal coiled-coil dimers (including both parallel and antiparallel coiled coils) as a hub for linking other components of a previously described synthetic molecular motor, the Tumbleweed. We use circular dichroism, analytical ultracentrifugation, dynamic light scattering, and disulfide rearrangement studies to demonstrate the ability of this six-peptide set to form the structure designed for the Tumbleweed motor. The successful formation of a suitable hub structure is both a test of the transferability of design rules for protein folding as well as an important step in the production of a synthetic protein-based molecular motor.

Trauma to the central and peripheral nervous systems often lead to serious morbidity. Current surgical methods for repairing or replacing such damage have limitations. Tissue engineering offers a potential alternative. Here we show that functionalized α-helical-peptide hydrogels can be used to induce attachment, migration, proliferation and differentiation of murine embryonic neural stem cells (NSCs). Specifically, compared with undecorated gels, those functionalized with Arg-Gly-Asp-Ser (RGDS) peptides increase the proliferative activity of NSCs; promote their directional migration; induce differentiation, with increased expression of microtubule-associated protein-2, and a low expression of glial fibrillary acidic protein; and lead to the formation of larger neurospheres. Electrophysiological measurements from NSCs grown in RGDS-decorated gels indicate developmental progress toward mature neuron-like behavior. Our data indicate that these functional peptide hydrogels may go some way toward overcoming the limitations of current approaches to nerve-tissue repair.

Iron-sulfur cluster proteins exhibit a range of physicochemical properties that underpin their functional diversity in biology, which includes roles in electron transfer, catalysis, and gene regulation. Transcriptional regulators that utilize iron-sulfur clusters are a growing group that exploit the redox and coordination properties of the clusters to act as sensors of environmental conditions including O2, oxidative and nitrosative stress, and metabolic nutritional status. To understand the mechanism by which a cluster detects such analytes and then generates modulation of DNA-binding affinity, we have undertaken a combined strategy of in vivo and in vitro studies of a range of regulators. In vitro studies of iron-sulfur cluster proteins are particularly challenging because of the inherent reactivity and fragility of the cluster, often necessitating strict anaerobic conditions for all manipulations. Nevertheless, and as discussed in this Account, significant progress has been made over the past decade in studies of O2-sensing by the fumarate and nitrate reduction (FNR) regulator and, more recently, nitric oxide (NO)-sensing by WhiB-like (Wbl) and FNR proteins. Escherichia coli FNR binds a [4Fe-4S] cluster under anaerobic conditions leading to a DNA-binding dimeric form. Exposure to O2 converts the cluster to a [2Fe-2S] form, leading to protein monomerization and hence loss of DNA binding ability. Spectroscopic and kinetic studies have shown that the conversion proceeds via at least two steps and involves a [3Fe-4S](1+) intermediate. The second step involves the release of two bridging sulfide ions from the cluster that, unusually, are not released into solution but rather undergo oxidation to sulfane (S(0)) subsequently forming cysteine persulfides that then coordinate the [2Fe-2S] cluster. Studies of other [4Fe-4S] cluster proteins that undergo oxidative cluster conversion indicate that persulfide formation and coordination may be more common than previously recognized. This remarkable feature suggested that the original [4Fe-4S] cluster can be restored using persulfide as the source of sulfide ion. We have demonstrated that only iron and a source of electrons are required to promote efficient conversion back from the [2Fe-2S] to the [4Fe-4S] form. We propose this as a novel in vivo repair mechanism that does not require the intervention of an iron-sulfur cluster biogenesis pathway. A number of iron-sulfur regulators have evolved to function as sensors of NO. Although it has long been known that the iron-sulfur clusters of many phylogenetically unrelated proteins are vulnerable to attack by NO, our recent studies of Wbl proteins and FNR have provided new insights into the mechanism of cluster nitrosylation, which overturn the commonly accepted view that the product is solely a mononuclear iron dinitrosyl complex (known as a DNIC). The major reaction is a rapid, multiphase process involving stepwise addition of up to eight NO molecules per [4Fe-4S] cluster. The major iron nitrosyl product is EPR silent and has optical characteristics similar to Roussin's red ester, [Fe2(NO)4(RS)2] (RRE), although a species similar to Roussin's black salt, [Fe4(NO)7(S)3](-) (RBS) cannot be ruled out. A major future challenge will be to clarify the nature of these species.

Protein engineering, chemical biology, and synthetic biology would benefit from toolkits of peptide and protein components that could be exchanged reliably between systems while maintaining their structural and functional integrity. Ideally, such components should be highly defined and predictable in all respects of sequence, structure, stability, interactions, and function. To establish one such toolkit, here we present a basis set of de novo designed α-helical coiled-coil peptides that adopt defined and well-characterized parallel dimeric, trimeric, and tetrameric states. The designs are based on sequence-to-structure relationships both from the literature and analysis of a database of known coiled-coil X-ray crystal structures. These give foreground sequences to specify the targeted oligomer state. A key feature of the design process is that sequence positions outside of these sites are considered non-essential for structural specificity; as such, they are referred to as the background, are kept non-descript, and are available for mutation as required later. Synthetic peptides were characterized in solution by circular-dichroism spectroscopy and analytical ultracentrifugation, and their structures were determined by X-ray crystallography. Intriguingly, a hitherto widely used empirical rule-of-thumb for coiled-coil dimer specification does not hold in the designed system. However, the desired oligomeric state is achieved by database-informed redesign of that particular foreground and confirmed experimentally. We envisage that the basis set will be of use in directing and controlling protein assembly, with potential applications in chemical and synthetic biology. To help with such endeavors, we introduce Pcomp, an on-line registry of peptide components for protein-design and synthetic-biology applications.

[Structure: see text] Protonation controls the location of a dual binding mode macrocycle in a [2]rotaxane. In the neutral form, amide-amide hydrogen bonds hold the macrocycle over a dipeptide residue; when the thread is protonated, polyether-ammonium cation interactions dominate and the macrocycle changes position.