Contrasting roles for two conserved arginines: Stabilizing flavin semiquinone or quaternary structure, in bifurcating electron transfer flavoproteins.

Bifurcating electron transfer flavoproteins (Bf ETFs) are important redox enzymes that contain two flavin adenine dinucleotide (FAD) cofactors, with contrasting reactivities and complementary roles in electron bifurcation. However, for both the "electron transfer" (ET) and the "bifurcating" (Bf) FADs, the only charged amino acid within 5 Å of the flavin is a conserved arginine (Arg) residue. To understand how the two sites produce different reactivities utilizing the same residue, we investigated the consequences of replacing each of the Arg residues with lysine, glutamine, histidine, or alanine. We show that absence of a positive charge in the ET site diminishes accumulation of the anionic semiquinone (ASQ) that enables the ET flavin to act as a single electron carrier, due to depression of the oxidized versus. ASQ reduction midpoint potential, E°OX/ASQ. Perturbation of the ET site also affected the remote Bf site, whereas abrogation of Bf FAD binding accelerated chemical modification of the ET flavin. In the Bf site, removal of the positive charge impaired binding of FAD or AMP, resulting in unstable protein. Based on pH dependence, we propose that the Bf site Arg interacts with the phosphate(s) of Bf FAD or AMP, bridging the domain interface via a conserved peptide loop ("zipper") and favoring nucleotide binding. We further propose a model that rationalizes conservation of the Bf site Arg even in non-Bf ETFs, as well as AMP's stabilizing role in the latter, and provides a mechanism for coupling Bf flavin redox changes to domain-scale motion.

Antihyperglycemic profile of erinidine isolated from Hunteria umbellata seed.

Water decoction made from the seed of Hunteria umbellata is widely used in the traditional management of diabetes mellitus by Nigerian herbalists, particularly, in the southwest region of the country. Recently, a new bisindole alkaloid, erinidine, was isolated but its antihyperglycemic profile remains largely un-investigated scientifically. This forms the basis for the current study which is primarily designed at investigating the antihyperglycemic profile of erinidine and other fractions in both in vitro and in vivo models of diabetes mellitus. In the present study, erinidine was isolated and purified using the earlier described methods and its antihyperglycemic potentials tested in in vitro models such as dipeptidylpeptidase (IV), glycogen phosphorylase, HIT-T15 cell insulin secretion, glucose uptake activity, aldose reductase assays and α-glucosidase inhibition assay testings. In addition, 50 mg/kg of erinidine and that of other fractions were evaluated in in vivo models of normal and chemically-induced hyperglycemic rats. Results showed that erinidine was a light yellow, amorphous solid with UV (CHCl3) λ max 256 nm, HRESIMS m/z 382.1881 [(M+H)(+)] (calculated for C22H26N4O2, 382.1876) and melting point of 230 °C. The in vitro study showed the antihyperglycemic action of erinidine to be weakly mediated via α-glucosidase inhibition mechanism as the results for other in vitro tests such as dipeptidylpeptidase (IV), glycogen phosphorylase, HIT-T15 cell insulin secretion, glucose uptake activity and aldose reductase assays were all negative. However, the in vivo results showed 50 mg/kg erinidine given per os to normal and alloxan-induced hyperglycemic rats to significantly (p<0.05, p<0.001) attenuate an increase in their post-absorptive blood glucose concentrations after 3 g/kg glucose loading in the rats, suggesting its antihyperglycemic mechanism to be via α-glucosidase inhibition. This result, although, further corroborated the in vitro findings but also suggests that erinidine needs to be biotransformed in vivo for its inhibitory activity on intestinal glucose absorption to become evident. Thus, the present study suggests erinidine to be the possible antihyperglycemic agent in Hunteria umbellata seed extract mediating its antihyperglycemic action via intestinal glucose uptake inhibition.

Redox tuning over almost 1 V in a structurally conserved active site: lessons from Fe-containing superoxide dismutase.

Metalloenzymes catalyze some of the most demanding reactions in biochemistry, thereby enabling organisms to extract energy from redox reactions and utilize inorganic starting materials such as N 2 and CH 4. Bound metal ions bring to enzymes greater chemical versatility and reactivity than would be possible from amino acids alone. However the host proteins must control this broad reactivity, activating the metal for the intended reaction while excluding the rest of its chemical repertoire. To this end, metalloproteins must control the metal ion reduction midpoint potential ( E m), because the E m determines what redox reactions are possible. We have documented potent redox tuning in Fe- and Mn-containing superoxide dismutases (FeSODs and MnSODs), and manipulated it to generate FeSOD variants with E ms spanning 900 mV (21 kcal/mol or 87 kJ/mol) with retention of overall structure. This achievement demonstrates possibilities and strategies with great promise for efforts to design or modify catalytic metal sites. FeSODs and MnSODs oxidize and reduce superoxide in alternating reactions that are coupled to proton transfer, wherein the metal site is believed to cycle between M3+ x OH- and M2+ x OH2 (M = Fe or Mn). Thus the E m reflects the ease both of reducing the metal ion and of protonating the coordinated solvent molecule. Moreover similar E ms are achieved by Fe-specific and Mn-specific SODs despite the very different intrinsic E(m)s of high-spin Fe3+/2+ and Mn3+/2+. We provide evidence that E(m) depression by some 300 mV can be achieved via a key enforced H-bond that appears able to disfavor proton acquisition by coordinated solvent. Based on 15N-nuclear magnetic resonance (NMR), stronger H-bond donation to coordinated solvent can explain the greater redox depression achieved by the Mn-specific SOD protein compared with the Fe-specific protein. Furthermore, by manipulating the strength and polarity of this one H-bond, with comparatively minor perturbation to active site atomic and electronic structure, we succeeded in raising the E m of FeSOD by more than 660 mV, apparently by a combination of promoting protonation of coordinated solvent and providing an energetically favorable source of a redox-coupled proton. These studies have combined the use of electron paramagnetic resonance (EPR), NMR, magnetic circular dichroism (MCD), and optical spectrophotometry to characterize the electronic structures of the various metal sites, with complementary density functional theoretical (DFT) calculations, NMR spectroscopy, and X-ray crystallography to define the protein structures and protonation states. Overall, we have generated structurally homologous Fe sites that span some 900 mV, and have demonstrated the enormous redox tuning accessible via the energies associated with proton transfer coupled to electron transfer. In this regard, we note the possible significance of coordinated solvent molecules in numerous biological redox-active metal sites besides that of SOD.

Second-sphere contributions to substrate-analogue binding in iron(III) superoxide dismutase.

A combination of spectroscopic and computational methods has been employed to explore the nature of the yellow and pink low-temperature azide adducts of iron(III) superoxide dismutase (N(3)-FeSOD), which have been known for more than two decades. Variable-temperature variable-field magnetic circular dichroism (MCD) data suggest that both species possess similar ferric centers with a single azide ligand bound, contradicting previous proposals invoking two azide ligands in the pink form. Complementary data obtained on the azide complex of the Q69E FeSOD mutant reveal that relatively minor perturbations in the metal-center environment are sufficient to produce significant spectral changes; the Q69E N(3)-FeSOD species is red in color at all temperatures. Resonance Raman (RR) spectra of the wild-type and Q69E mutant N(3)-FeSOD complexes are consistent with similar Fe-N(3) units in all three species; however, variations in energies and relative intensities of the RR features associated with this unit reveal subtle differences in (N(3)(-))-Fe(3+) bonding. To understand these differences on a quantitative level, density functional theory and semiempirical INDO/S-CI calculations have been performed on N(3)-FeSOD models. These computations support our model that a single azide ligand is present in all three N(3)-FeSOD adducts and suggest that their different appearances reflect differences in the Fe-N-N bond angle. A 10 degrees increase in the Fe-N-N bond angle is sufficient to account for the spectral differences between the yellow and pink wild-type N(3)-FeSOD species. We show that this bond angle is strongly affected by the second coordination sphere, which therefore might also play an important role in orienting incoming substrate for reaction with the FeSOD active site.