Cupredoxins are small proteins that contain type I copper centers, which are ubiquitous in nature. They function as electron transfer shuttles between proteins. This review of the structure and properties of native cupredoxins, and those modified by site-directed mutagenesis, illustrates how these proteins may have evolved to specifically bind copper, develop recognition sites for specific redox partners, tune redox potential for a particular function, and allow for efficient electron transfer through the protein matrix. This is relevant to the general understanding of the roles of metals in energy metabolism, respiration and photosynthesis.

Respiration, photosynthesis and metabolism require the transfer of electrons through and between proteins over relatively long distances. It is critical that this electron transfer (ET) occur with specificity to avoid cellular damage, and at a rate which is sufficient to support the biological activity. A multi-step hole hopping mechanism could, in principle, enhance the efficiency of long range ET through proteins as it does in organic semiconductors. To explore this possibility, two different ET reactions that occur over the same distance within the protein complex of the diheme enzyme MauG and different forms of methylamine dehydrogenase (MADH) were subjected to kinetic and thermodynamic analysis. An ET mechanism of single-step direct electron tunneling from diferrous MauG to the quinone form of MADH is consistent with the data. In contrast, the biosynthetic ET from preMADH, which contains incompletely synthesized tryptophan tryptophylquinone, to the bis-Fe(IV) form of MauG is best described by a two-step hole hopping mechanism. Experimentally-determined values of ET distance matched the distances determined from the crystal structure that would be expected for single-step tunneling and multi-step hopping, respectively. Experimentally-determined relative values of electronic coupling (HAB) for the two reactions correlated well with the relative HAB values predicted from computational analysis of the structure. The rate of the hopping-mediated ET reaction is also ten-fold greater than that of the single-step tunneling reaction despite having a smaller overall driving force for the reaction. These data provide insight into how the intervening protein matrix and redox potentials of the electron donor and acceptor determine whether the ET reaction proceeds via single-step tunneling or multi-step hopping.

The effects on the structure and function of amicyanin of replacing the axial methionine ligand of the type 1 copper site with leucine have been characterized. The crystal structures of the oxidized and reduced forms of the protein reveal that the copper site is now tri-coordinate with no axial ligand, and that the copper coordination distances for the two ligands provided by histidines are significantly increased. Despite these structural changes, the absorption and EPR spectra of M98L amicyanin are only slightly altered and still consistent with that of a typical type 1 site. The oxidation-reduction midpoint potential (E m ) value becomes 127 mV more positive as a consequence of the M98L mutation, most likely due to increased hydrophobicity of the copper site. The most dramatic effect of the mutation was on the electron transfer (ET) reaction from reduced M98L amicyanin to cytochrome c-551i within the protein ET complex. The rate decreased 435-fold, which was much more than expected from the change in E m value. Examination of the temperature dependence of the ET rate (k ET ) revealed that the mutation caused a 13.6 fold decrease in the electronic coupling (H AB ) for the reaction. A similar decrease was predicted from a comparative analysis of the crystal structures of reduced M98L and native amicyanins. The most direct route of ET for this reaction is through the Met98 ligand. Inspection of the structures suggests that the major determinant of the large decrease in the experimentally determined values of H AB and k ET is the increased distance from the copper to the protein within the type 1 site of M98L amicyanin.Copper proteins are common in nature and involved in a variety of biological processes such as respiration, photosynthesis, and redox reactions critical to metabolism (1). The copper centers are classified according to their spectroscopic properties as type 1, type 2 or type 3. Type 1 copper sites are found in a wide range of electron transfer (ET) proteins, including amicyanin and azurin in bacteria, plastocyanin in plants, and multicopper oxidases such as NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2010 October 6. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript fungal laccase and human ceruloplasmin (1). The type 1 copper site of cupredoxins consists of three relatively strong equatorial ligands, nitrogens of two histidines and sulfur of a cysteine, forming a trigonal plane. A weaker fourth axial ligand is usually, but not always, provided by a sulfur of a methionine (2). Azurin is atypical in that additional coordination to the copper is provided by the backbone carbonyl O of a Gly, giving that copper site a trigonal bipyramidal geometry. In amicyanin from P. denitrificans the three strong equatorial copper ligands are provided by residues His53, His95 and Cys92, and the weak axial ligand is provided by Met98 (3).Amicyanin (4) serves as a mediator of ET from methylamine dehydrogenase (MADH) (5) to cytochrome c-551i (6)....

M98Q amicyanin is isolated with zinc bound to its type 1 copper-binding site. The influence of the axial ligand of the type 1 copper site on metal specificity is strongest prior to the completion of protein folding and adoption of the final type 1 site geometry. The preference for zinc over copper correlated with the selectivity of apoamicyanin in vitro in the partially folded, rather than the completely folded state. These results suggest that metal incorporation in vivo occurs during protein folding in the periplasm and not to a preformed type 1 site.

Mitochondrial metabolism plays a crucial role in insulin resistance and insulin secretion in type 2 diabetes mellitus (T2D). Some studies have focused on how Cassia tora extracts affect insulin resistance and hyperglycemia. However, the effects of Cassia tora extracts on mitochondrial dysfunction associated with insulin secretion have not been well explained. In this study, well-known effective compounds extracted from Cassia tora using 70% ethanol were administered to a high-fat diet (HFD) fed mouse to examine the effects of Cassia tora ethanolic extracts (CSEE) on mitochondrial dysfunction in the pancreas. Furthermore, we examined how CSEE regulates the basal mechanism of insulin secretion through mitochondrial functions. Our experimental data suggest that pancreatic mitochondrial metabolism in HFD mice is enhanced to compensate for constrained glucose consumption. HFD-fed mice treated with CSEE showed improved pancreatic mitochondrial functions resulting in alleviation of insulin resistance at target tissue as well as basal hyperinsulinemia.

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Tobacco etch virus (TEV) protease is a 27‐kDa catalytic domain of the polyprotein nuclear inclusion a (NIa) in TEV, which recognizes the specific amino acid sequence ENLYFQG/S and cleaves between Q and G/S. Despite its substrate specificity, its use is limited by its autoinactivation through self‐cleavage and poor solubility during purification. It was previously reported that T17S/N68D/I77V mutations improve the solubility and yield of TEV protease and S219 mutations provide protection against self‐cleavage. In this study, we isolated TEV proteases with S219N and S219V mutations in the background of T17S, N68D, and I77V without the inclusion body, and measured their enzyme kinetics. The kcat of two isolated S219N and S219V mutants in the background of T17S, N68D, and I77V mutations was highly increased compared to that of the control, and S219N was twofold faster than S219V without Km change. This result indicates that combination of these mutations can further enhance TEV activity.

The diheme enzyme MauG catalyzes a six-electron oxidation required for posttranslational modification of a precursor of methylamine dehydrogenase (preMADH) to complete the biosynthesis of its protein-derived tryptophan tryptophylquinone (TTQ) cofactor. One heme is low-spin with ligands provided by His205 and Tyr294, and the other is high-spin with a ligand provided by His35. The side chain methyl groups of Thr67 and Leu70 are positioned at a distance of 3.4 Å on either side of His35, maintaining a hydrophobic environment in the proximal pocket of the high-spin heme and restricting the movement of this ligand. Mutation of Thr67 to Ala in the proximal pocket of the high-spin heme prevented reduction of the low-spin heme by dithionite, yielding a mixed-valent state. The mutation also enhanced the stabilization of the charge-resonance-transition of the high-valent bis-FeIV state that is generated by addition of H2O2. The rates of electron transfer from TTQ biosynthetic intermediates to the high-valent form of T67A MauG were similar to that of wild-type MauG. These results are compared to those previously reported for mutation of residues in the distal pocket of the high-spin heme that also affected the redox properties and charge resonance transition stabilization of the high-valent state of the hemes. However, given the position of residue 67, the structure of the variant protein and the physical nature of the T67A mutation, the basis for the effects of the T67A mutation must be different from those of the mutations of the residues in the distal heme pocket.