Functionalised peptide hydrogel for the delivery of cardiac progenitor cells.

Heart failure (HF) remains one of the leading causes of death worldwide; most commonly developing after myocardial infarction (MI). Since adult cardiomyocytes characteristically do not proliferate, cells lost during MI are not replaced. As a result, the heart has a limited regenerative capacity. There is, therefore, a need to develop novel cell-based therapies to promote the regeneration of the heart after MI. The delivery and retention of cells at the injury site remains a significant challenge. In this context, we explored the potential of using an injectable, RGDSP-functionalised self-assembling peptide - FEFEFKFK - hydrogel as scaffold for the delivery and retention of rat cardiac progenitor cells (CPCs) into the heart. Our results show that culturing CPCs in vitro within the hydrogel for one-week promoted their spontaneous differentiation towards adult cardiac phenotypes. Injection of the hydrogel on its own, or loaded with CPCs, into the rat after injury resulted in a significant reduction in myocardial damage and left ventricular dilation.

Reduction midpoint potentials of bifurcating electron transfer flavoproteins.

Recently, a variety of enzymes have been found to accept electrons from NAD(P)H yet reduce lower-potential carriers such as ferredoxin and flavodoxin semiquinone, in apparent violation of thermodynamics. The reaction is favorable overall, however, because these enzymes couple the foregoing endergonic one-electron transfer to exergonic transfer of the other electron from each NAD(P)H, in a process called "flavin-based electron bifurcation." The reduction midpoint potentials (E°s) of the multiple flavins in these enzymes are critical to their mechanisms. We describe methods we have found to be useful for measuring each of the E°s of each of the flavins in bifurcating electron transfer flavoproteins.

In vivo characterisation of a therapeutically relevant self-assembling 18 F-labelled ß-sheet forming peptide and its hydrogel using positron emission tomography.

Positron emission tomography (PET) and fluorescence labelling have been used to assess the pharmacokinetics, biodistribution and eventual fate of a hydrogel-forming nonapeptide, FEFKFEFKK (F9), in healthy mice, using 18 F-labelled and fluorescein isothiocyanate (FITC)-labelled F9 analogues. F9 was site-specifically radiolabelled with 2-[18 F]fluoro-3-pyridinecarboxaldehyde ([18 F]FPCA) via oxime bond formation. [18 F]FPCA-F9 in vivo fate was evaluated both as a solution, following intravenous administration, and as a hydrogel when subcutaneously injected. The behaviour of FITC-F9 hydrogel was assessed following subcutaneous injection. [18 F]FPCA-F9 demonstrated high plasma stability and primarily renal excretion; [18 F]FPCA-F9 when in solution and injected into the bloodstream displayed prompt bladder uptake (53.4 ± 16.6 SUV at 20 minutes postinjection) and rapid renal excretion, whereas [18 F]FPCA-F9 hydrogel, formed by co-assembly of [18 F]FPCA-F9 monomer with unfunctionalised F9 peptide and injected subcutaneously, showed gradual bladder accumulation of hydrogel fragments (3.8 ± 0.4 SUV at 20 minutes postinjection), resulting in slower renal excretion. Gradual disaggregation of the F9 hydrogel from the site of injection was monitored using FITC-F9 hydrogel in healthy mice (60 ± 3 over 96 hours), indicating a biological half-life between 1 and 4 days. The in vivo characterisation of F9, both as a gel and a solution, highlights its potential as a biomaterial.

Peptide hydrogel in vitro non-inflammatory potential.

Peptide-based hydrogels have attracted significant interest in recent years as these soft, highly hydrated materials can be engineered to mimic the cell niche with significant potential applications in the biomedical field. Their potential use in vivo in particular is dependent on their biocompatibility, including their potential to cause an inflammatory response. In this work, we investigated in vitro the inflammatory potential of a ß-sheet forming peptide (FEFEFKFK; F: phenylalanine, E: glutamic acid; K: lysine) hydrogel by encapsulating murine monocytes within it (3D culture) and using the production of cytokines, IL-ß, IL-6 and TNFα, as markers of inflammatory response. No statistically significant release of cytokines in our test sample (media + gel + cells) was observed after 48 or 72 h of culture showing that our hydrogels do not incite a pro-inflammatory response in vitro. These results show the potential biocompatibility of these hydrogels and therefore their potential for in vivo use. The work also highlighted the difference in monocyte behaviour, proliferation and morphology changes when cultured in 2D vs. 3D. © 2016 The Authors Journal of Peptide Science published by European Peptide Society and John Wiley & Sons Ltd.

Self-assembling peptide/thermoresponsive polymer composite hydrogels: effect of peptide-polymer interactions on hydrogel properties.

We have investigated the effect of doping the self-assembling octapeptide FEFEFKFK (F, phenylalanine; E, glutamic acid; K, lysine) hydrogels with various amounts of thermoresponsive conjugate of FEFEFKFK and poly(N-isopropylacrylamide) (PNIPAAm) in order to create novel hydrogels. The samples were characterized using a range of techniques including microdifferential scanning calorimetry (µDSC), oscillatory rheology, transmission electron microscopy (TEM), atomic force microscopy (AFM), and small angle neutron scattering (SANS). The peptide from the conjugate was shown to be incorporated into the peptide fiber, resulting in the polymer being anchored to the peptide fiber. The conjugation of the polymer to the peptide and its anchoring to the peptide fibers did not affect its lower critical solution temperature (LCST). On the other hand, it did result in a decrease in the LCST enthalpy and a significant increase in the G' of the hydrogels, suggesting the presence of hydrogen bond interactions between the peptide and the polymer. As a result, the polymer was found to adopt a fibrillar arrangement tightly covering the peptide fiber. The polymer was still found to go through a conformational change at the LCST, suggesting that it collapses onto the peptide fiber. On the other hand, the fibrillar network was found to be mainly unaffected by the polymer LCST. These changes at the LCST were also found to be fully reversible. The nature of the interaction between the polymer and the peptide was shown to have a significant effect on the conformation adopted by the polymer around the fibers and the mechanical properties of the hydrogels.

Effect of peptide and guest charge on the structural, mechanical and release properties of ß-sheet forming peptides.

The effect of peptide charge on the self-assembly, gelation behavior, and model drug release profiles has been explored here for three octa-peptides, VEVKVEVK (VEK2), VKVKVEVK (VEK3), and VEVEVKVE (VEK1), that carry a net charge of 0, +2, and -2 at neutral pH, respectively. Transparent, self-supporting hydrogels were found to form above a critical concentration when the peptide charge modulus was >1 and this was independent of the sign of the charge. TEM, SAXS, and shear rheology revealed that there were no differences in hydrogel structure or mechanical properties when the peptides were at the same concentration and carried the same charge modulus. All peptides were found to form dense fibrillar networks formed by ß-sheet rich single fibers where lateral aggregation of the fibers occurred and increased with decreasing charge modulus. Such behavior was found to correlate with an increase in hydrogel mechanical properties, demonstrating that fiber lateral aggregation is inextricably linked with the mechanical properties of these hydrogels. Two hydrophilic model drug molecules, namely napthol yellow (NY) and martius yellow (MY), were subsequently incorporated within the VEK1 and VEK3 hydrogels at pH 7 and although they did not effect the self-assembly of the peptide at a molecular level, they did effect the level of lateral fiber aggregation observed and, therefore, the mechanical properties of the hydrogels. The release of each molecule from the hydrogels was monitored over time and shown to be controlled by Fickian diffusion where the diffusion rate, D, was dependent on the ratio between the overall effective charges carried by the peptide, i.e., the fibrillar network, and the overall charges carried by the guest molecules, but independent from the hydrogel concentration and mechanical properties within the ranges investigated. This work highlights the possibility of controlling the rate of release of small drug molecules by manipulating the charges on the guest molecules as well as the charged state of the self-assembling peptide.

Carbon-centered radicals in isolated soy proteins.

Solid-state electron paramagnetic resonance (EPR) spectroscopy of commercial samples of isolated soy proteins (ISP) revealed a symmetrical free-radical signal typical of carbon-centered radicals (g= 2.005) ranging from 2.96 x 10(14) to 6.42 x 10(14) spins/g. The level of free radicals in ISP was 14 times greater than similar radicals in sodium caseinate, 29 times greater than egg albumin, and about 100 times greater levels than casein. Nine soy protein powdered drink mixes contained similar types of free radicals up to 4.10 x 10(15) spins/g of drink mix, or up to 6.4 times greater than the highest free-radical content found in commercial ISP. ISP samples prepared in the laboratory contained trapped radicals similar to the levels in commercial ISP samples. When ISP was hydrated in 2.3 mM sodium erythorbate or 8.3 mM L-cysteine, frozen and dried, the level of trapped free radicals increased by about 17- and 19-fold, respectively. The ESR spectrum of defatted soybean flakes contained overlapping signals from the primary free-radical peak (g= 2.005) and a sextet pattern typical of manganese-II. The manganese signal was reduced in the laboratory ISP and very weak in the commercial ISP.

Thermoreversible lysozyme hydrogels: properties and an insight into the gelation pathway.

The gelation behaviour of aqueous solutions of hen egg white lysozyme (HEWL) in the presence of 20 mM DTT in the concentration range 0.7 to 4.0 mM has been investigated using microDSC, FTIR, cryoTEM, SANS and oscillatory rheology. The macroscopic critical gelation concentration, Cgel, was found to be ∼ 3.0 mM. The disruption of the disulfide bonds by the DTT and the destabilisation of the protein were found to be a prerequisite for the formation of ß-sheet rich fibrils under the mild conditions used in this work. Using our methodology the hydrogels obtained have a pH of 7, hence are suitable for cell culture, and are also thermoreversible. The hydrogel melting temperature was found to increase with increasing concentration and a similar structure was observed across the concentration range investigated. Our results suggest these systems are composed of a well defined regular network where single ß-sheet rich fibrils (∼ 3 nm diameter) form initially, then two of these fibrils associate two-by-two to form junctions (∼ 6 nm diameter) and then on cooling further aggregate to form larger bundles of fibres. The network mesh size was found to decrease with increasing concentration. Our results suggest that below Cgel small unconnected gel-like aggregates exist that have a similar structure to the hydrogels obtained above Cgel. Using our data we propose a model for the denaturation and gelation behaviour of our system. During the first heating an α-helix to ß-sheet molecular transition for the protein conformation occurs resulting in ß-sheet rich fibrils forming through the self-assembly of ß-sheet rich denaturated proteins. At high temperature the solution contains ß-sheet rich fibrils with dissolved protein. On cooling an increase in the amount of ß-sheet was observed via FTIR suggesting that as the temperature is decreased more and more protein forms ß-sheet rich fibrils. At the gelation temperature these fibrils associate two-by-two to form the network junctions resulting in the macroscopic gelation of the sample. Our results suggest the network junctions are formed via specific hydrophobic interactions. The hydrogels elastic modulus was found to scale as C2.45 for C > Cgel.

Host-guest study of left-handed polyproline II helix formation.

The importance of the left-handed polyproline II (PPII) helical conformation has recently become apparent. This conformation generally is involved in two important functions: protein-protein interactions and structural integrity. PPII helices play vital roles in a variety of processes including signal transduction, transcription, and cell motility. Proline-rich regions of sequence are often assumed to adopt this structure. Remarkably, little is known about the physical determinants of this secondary structure type. In this study, we have explored the formation of PPII helices by a short poly(proline) peptide. In addition, the results from experiments used to determine the propensities for apolar residues, plus glycine, asparagine, and glutamine, to adopt this structure in a poly(proline)-based host peptide are reported here. Proline possesses the highest intrinsic propensity, with glutamine, alanine, and glycine having surprisingly high propensities. beta-Branched residues possess the lowest propensities of the residues examined. It is postulated that propensities possessed by apolar residues are due in part to peptide-solvent interactions, and that the remarkably high propensity possessed by glutamine may be due to a side chain to backbone hydrogen bond. These data are the first step toward a molecular understanding of the formation of this important, and yet little studied, secondary structure.

Novel insights into the basis for Escherichia coli superoxide dismutase's metal ion specificity from Mn-substituted FeSOD and its very high E(m).

Fe and Mn are both entrained to the same chemical reaction in apparently superimposable superoxide dismutase (SOD) proteins. However, neither Fe-substituted MnSOD nor Mn-substituted FeSOD is active. We have proposed that the two SOD proteins must apply very different redox tuning to their respective metal ions and that tuning appropriate for one metal ion results in a reduction potential (E(m)) for the other metal ion that is either too low (Fe) or too high (Mn) [Vance and Miller (1998) J. Am. Chem. Soc. 120, 461-467]. We have demonstrated that this is true for Fe-substituted MnSOD from Escherichia coli and that this metal ion-protein combination retains the ability to reduce but not oxidize superoxide. We now demonstrate that the corollary is also true: Mn-substituted FeSOD [Mn(Fe)SOD] has a very high E(m). Specifically, we have measured the E(m) of E. coli MnSOD to be 290 mV vs NHE. We have generated Mn(Fe)SOD and find that Mn is bound in an environment similar to that of the native (Mn)SOD protein. However, the E(m) is greater than 960 mV vs NHE and much higher than MnSOD's E(m) of 290 mV. We propose that the different tuning stems from different hydrogen bonding between the proteins and a molecule of solvent that is coordinated to the metal ion in both cases. Because a proton is taken up by SOD upon reduction, the protein can exert very strong control over the E(m), by modulating the degree to which coordinated solvent is protonated, in both oxidation states. Thus, coordinated solvent molecules may have widespread significance as "adapters" by which proteins can control the reactivity of bound metal ions.

Quantitative structure-activity relationships in two-electron reduction of nitroaromatic compounds by Enterobacter cloacae NAD(P)H:nitroreductase.

Enterobacter cloacae NAD(P)H:nitroreductase (NR; EC 1.6.99.7) catalyzes the reduction of a series of nitroaromatic compounds with steady-state bimolecular rate constants (kcat/Km) ranging from 10(4) to 10(7) M(-1) s(-1). In agreement with a previously proposed scheme of two-step four-electron reduction of nitroaromatics by NR (Koder, R. L., and Miller, A.-F. (1998) Biochim. Biophys. Acta 1387, 395-405), 2 mol NADH per mole mononitrocompound were oxidized. An oxidation of excess NADH by polinitrobenzenes, including explosives 2,4,6-trinitrotoluene (TNT) and 2,4,6-trinitrophenyl-N-methylnitramine (tetryl), has been observed as a slower secondary process, accompanied by O2 consumption. This type of "redox cycling" was not related to reactions of nitroaromatic anion-radicals, but was caused by the autoxidation of relatively stable reaction products. The initial reduction of tetryl and other polinitrophenyl-N-nitramines by E. cloacae NR was analogous to a two-step four-electron reduction mechanism of TNT and other nitroaromatics. The logs kcat/Km of all the compounds examined exhibited parabolic dependence on their enthalpies of single-electron or two-electron (hydride) reduction, obtained by quantum mechanical calculations. This type of quantitative structure-activity relationship shows that the reactivity of nitroaromatics towards E. cloacae nitroreductase depends mainly on their hydride accepting properties, but not on their particular structure, and does not exclude the possibility of multistep hydride transfer.

Retro-nitroreductase, a putative evolutionary precursor to Enterobacter cloacae strain 96-3 nitroreductase.

Enterobacter cloacae strain 96-3 nitroreductase (NR) is a homodimeric flavoenzyme that catalyzes the pyridine nucleotide-dependent four-electron reduction of a variety of nitroaromatic compounds, including the explosives TNT (2,4,6-trinitrotoluene), RDX (1,3,5-trinitro-1,3,5-triazine), tetryl (2,4,6-trinitrophenyl-N-methylnitramine), and pentryl (2,4,6-trinitrophenyl-N-nitroaminoethylnitrate). The enzyme was initially characterized by Bryant et al. from a strain of Enterobacter that had been isolated from a weapons dump in La Jolla, CA. The enzyme displays a catalytic efficiency for nitroreduction at least 10-fold higher than that of several highly homologous bacterial nitroreductases and has long been thought to have evolved to be a more efficient nitroreductase due to the high nitroaromatic compound concentrations in its environment. We report the cloning and biochemical characterization of a nitroreductase gene from a clinical isolate of Enterobacter cloacae, a strain that presumably had not encountered high concentrations of nitroaromatics. The new enzyme, which we term retro-nitroreductase, had an amino acid sequence 96.7% identical to NR, and most differences are relatively conservative. The catalytic efficiency of the new enzyme is twofold less than that of NR for the oxidation of NADH and is not significantly different from the value observed for NR for the reduction of dinitrobenzyl alcohol. We conclude that NR has not significantly evolved to be a more efficient nitroreductase as a result of its environment, and the relatively high catalytic activity of the enzyme is a general property of Enterobacter cloacae nitroreductases.

Amino acid-specific isotopic labeling and active site NMR studies of iron(II)- and iron(III)-superoxide dismutase from Escherichia coli.

We have developed and employed multiple amino acid-specific isotopic labeling schemes to obtain definitive assignments for active site 1H NMR resonances of iron(II)- and iron(III)-superoxide dismutase (Fe(II)SOD and Fe(III)SOD) from Escherichia coli. Despite the severe relaxivity of high-spin Fe(III), we have been able to assign resonances to ligand His' delta1 protons near 100 ppm, and beta and alpha protons collectively between 20 and 50 ppm, in Fe(III)SOD. In the reduced state, we have assigned all but 7 ligand protons, in most cases residue-specifically. A pair of previously unreported broad resonances at 25.9 and 22.1 ppm has been conclusively assigned to the beta protons of Asp 156, superseding earlier assignments (Ming et al. (1994) Inorg. Chem., 33, 83-87). We have exploited higher temperatures to resolve previously unobserved ortho-like ligand His proton resonances, and specific isotopic labeling to distinguish between the possibilities of 82 and epsilon1 protons. These are the closest protein protons to Fe(II) and therefore they have the broadest (approximately 4,000 Hz) and most difficult to detect resonances. Our assignments permit interpretation of temperature dependences of chemical shifts, pH dependences and H/D exchange rates in terms of a hydrogen bond network and the Fe(II) electronic state. Interestingly, Fe(II)SOD's axial His ligand chemical shifts are similar to those of the axial His ligand of Rhodopseudomonas palustris cytochrome c' (Bertini et al. (1988) Inorg. Chem., 37, 4814-4821 ) suggesting that Fe(II)SOD's equatorial His2Asp- ligation is able to reproduce some of the electronic, and thus possibly chemical, properties of heme coordination for Fe2+.

Mutational and spectroscopic studies of the significance of the active site glutamine to metal ion specificity in superoxide dismutase.

We are addressing the puzzling metal ion specificity of Fe- and Mn-containing superoxide dismutases (SODs) [see C.K.Vance, A.-F. Miller. J. Am. Chem. Soc. 120(3) (1998) 461-467]. Here, we test the significance to activity and active site integrity of the Gln side chain at the center of the active site hydrogen bond network. We have generated a mutant of MnSOD with the active site Gln in the location characteristic of Fe-specific SODs. The active site is similar to that of MnSOD when Mn2+, Fe3+ or Fe2+ are bound, based on EPR and NMR spectroscopy. However, the mutant's Fe-supported activity is at least 7% that of FeSOD, in contrast to Fe(Mn)SOD, which has 0% of FeSOD's activity. Thus, moving the active site Gln converts Mn-specific SOD into a cambialistic SOD and the Gln proves to be important but not the sole determinant of metal-ion specificity. Indeed, subtle differences in the spectra of Mn2+, Fe3+ and 1H in the presence of Fe2+ distinguish the G77Q, Q146A mut-(Mn)SOD from WT (Mn)SOD, and may prove to be correlated with metal ion activity. We have directly observed the side chain of the active site Gln in Fe2+ SOD and Fe2+ (Mn)SOD by 15N NMR. The very different chemical shifts indicate that the active site Gln interacts differently with Fe2+ in the two proteins. Since a shorter distance from Gln to Fe and stronger interaction with Fe correlate with a lower Em in Fe(Mn)SOD, Gln has the effect of destabilizing additional electron density on the metal ion. It may do this by stabilizing OH- coordinated to the metal ion.

Bone morphogenetic protein-9. An autocrine/paracrine cytokine in the liver.

Bone morphogenetic proteins (BMPs) occupy important roles during development serving to direct cells through specific differentiation programs. While several BMPs are essential for embryonic viability, their significance in mediating intercellular communication in the context of adult organ systems remains largely unknown. In the adult rat we characterized the tissue- and cell-specific transcription and translation of BMP-9. Utilizing a ribonuclease protection assay, we determined that in the adult animal, BMP-9 expression occurs predominantly in the liver. Furthermore, we determined that the non-parenchymal cells of the liver, i.e. endothelial, Kupffer, and stellate cells, are the major sources of this message. Western analyses corroborate the ribonuclease protection assay results, confirming that LEC and KC contain an abundance of immunoreactive BMP-9. Using [(125)I]BMP-9, a receptor with specific binding affinity for BMP-9 was characterized in primary cultures of hepatic endothelial cells and Kupffer cells. BMP-9 binding to these cell types was observed to be fully reversible and highly specific for this ligand. Additionally, we demonstrate that BMP-9 is specifically internalized upon binding to its receptor. This may represent a novel BMP receptor and is the first to be characterized in primary cultures of mature liver non-parenchymal cells. Our results depict BMP-9 as a potential autocrine/paracrine mediator in the hepatic reticuloendothelial system.

Two-electron reduction of nitroaromatic compounds by Enterobacter cloacae NAD(P)H nitroreductase: description of quantitative structure-activity relationships.

Enterobacter cloacae NAD(P)H:nitroreductase catalyzes the reduction of a series of nitroaromatic compounds with steady-state bimolecular rate constants (kcat/Km) ranging from 10(4) M(-1) s(-1) to 10(7) M(-1) s(-1), and oxidizing 2 moles NADH per mole mononitrocompound. Oxidation of excess NADH by polynitrobenzenes including explosives 2,4,6-trinitrotoluene (TNT) and 2,4,6-trinitrophenyl-N-methylnitramine (tetryl), has been observed as a slower secondary process, accompanied by O2 consumption. This type of 'redox cycling' was not related to reactions of nitroaromatic anion-radicals, but was caused by the autoxidation of relatively stable reaction products. The logs kcat/Km of all the compounds examined exhibited parabolic dependence on their enthalpies of single-electron- or two-electron (hydride) reduction, obtained by quantum mechanical calculations. This type of quantitative structure-activity relationships shows that the reactivity of nitroaromatics towards E. cloacae nitroreductase depends mainly on their hydride accepting properties, but not on their particular structure, and does not exclude the possibility of multistep hydride transfer.

Steady-state kinetic mechanism, stereospecificity, substrate and inhibitor specificity of Enterobacter cloacae nitroreductase.

Enterobacter cloacae nitroreductase (NR) is a flavoprotein which catalyzes the pyridine nucleotide-dependent reduction of nitroaromatics. Initial velocity and inhibition studies have been performed which establish unambiguously a ping-pong kinetic mechanism. NADH oxidation proceeds stereospecifically with the transfer of the pro-R hydrogen to the enzyme and the amide moiety of the nicotinamide appears to be the principal mediator of the interaction between NR and NADH. 2,4-Dinitrotoluene is the most efficient oxidizing substrate examined, with a kcat/KM an order of magnitude higher than those of p-nitrobenzoate, FMN, FAD or riboflavin. Dicoumarol is a potent inhibitor competitive vs. NADH with a Ki of 62 nM. Several compounds containing a carboxyl group are also competitive inhibitors vs. NADH. Yonetani-Theorell analysis of dicoumarol and acetate inhibition indicates that their binding is mutually exclusive, which suggests that the two inhibitors bind to the same site on the enzyme. NAD+ does not exhibit product inhibition and in the absence of an electron acceptor, no isotope exchange between NADH and 32P-NAD+ could be detected. NR catalyzes the 4-electron reduction of nitrobenzene to hydroxylaminobenzene with no optically detectable net formation of the putative two-electron intermediate nitrosobenzene.

Overexpression, isotopic labeling, and spectral characterization of Enterobacter cloacae nitroreductase.

Bacterial nitroreductases have generated much interest recently due to their central roles in both nitroaromatic bioremediation and nitroaromatic toxicity, mutagenicity, and carcinogenicity. Enterobacter cloacae nitroreductase (NR) has been subcloned into the pET overexpression system and purified to homogeneity via a four-step procedure resulting in a final yield of 65.7 mg per liter. Overexpression in minimal media containing 15NH4Cl as the sole source of nitrogen yielded 37.6 mg per liter of homogenous NR containing > 99 atom % 15N. A series of melting curves generated under a variety of solvent conditions established the optimal conditions for NR stability as pH 7.5, low ionic strength phosphate buffer. A two-dimensional 1H-15N heteronuclear single quantum coherence nuclear magnetic resonance spectrum demonstrates this enzyme to be amenable to study by high-resolution multidimensional NMR in combination with amino-acid-specific isotopic labeling. Optical spectra of the purified enzyme suggest that the noncovalently bound flavin mononucleotide cofactor binds in a hydrophobic environment and is in the neutral and anionic protonation states in the oxidized and two-electron reduced oxidation states, respectively. NR exhibits a novel visible region circular dichroism spectrum which has a small distinct negative band at 366 nm and a large positive ellipticity at 454 nm with a shoulder centered at 480 nm.

Spectroscopic comparisons of the pH dependencies of Fe-substituted (Mn)superoxide dismutase and Fe-superoxide dismutase.

We have compared the active sites of Escherichia coli Fe-substituted (Mn)superoxide dismutase [Fe-sub-(Mn)SOD] and Fe-SOD to elucidate the basis for the inactivity of Fe-sub-(Mn)SOD, despite its apparent similarity to Fe-SOD. The active site of (reduced) Fe2+-sub-(Mn)SOD is qualitatively similar to that of native Fe2+-SOD, indicating similar active site structures and coordination environments for Fe2+. Its nativelike pK is indicative of nativelike local electrostatics, and consistent with Fe2+-sub-(Mn)SOD's retention of ability to reduce O2*- [Vance and Miller (1998) J. Am. Chem. Soc. 120(3), 461-467]. The active site of (oxidized) Fe3+-sub-(Mn)SOD differs from that of Fe3+-SOD with respect to the EPR signals produced at both neutral and high pH, indicating different coordination environments for Fe3+. Although Fe3+-sub-(Mn)SOD binds the small anions N3- and F-, the KD for N3- is tighter than that of Fe3+-SOD, suggesting that the (Mn)SOD protein favors anion binding more than does the (Fe)SOD protein. The EPR spectral consequences of binding F- are reminiscent of those observed upon binding the first F- to Fe3+-SOD, but the EPR spectrum obtained upon binding N3- is different, consistent with crystallographic observation of a different binding mode for N3- in Thermus thermophilus Mn-SOD than Fe-SOD [Lah, M., et al. (1995) Biochemistry 34, 1646-1660]. We find a pK of 8.5 to be associated with dramatic changes in the EPR spectrum. In addition, we confirm the pK between 6 and 7 that has previously been reported based on changes in the optical signal and N3- binding [Yamakura, F., et al. (1995) Eur. J. Biochem. 227, 700-706]. However, this latter pK appears to be associated with much subtler changes in the EPR spectrum. The non-native pKs observed in Fe3+-sub-(Mn)SOD and the differences in the Fe3+ coordination indicated by the EPR spectra are consistent with Fe3+-sub-(Mn)SOD's inability to oxidize O2*- and suggest that its low E degrees is due to perturbation of the oxidized state.