In Vitro Demonstration of Dual Light-Driven Na⁺/H⁺ Pumping by a Microbial Rhodopsin.

A subfamily of rhodopsin pigments was recently discovered in bacteria and proposed to function as dual-function light-driven H(+)/Na(+) pumps, ejecting sodium ions from cells in the presence of sodium and protons in its absence. This proposal was based primarily on light-induced proton flux measurements in suspensions of Escherichia coli cells expressing the pigments. However, because E.

Cation-Specific Conformations in a Dual-Function Ion-Pumping Microbial Rhodopsin.

A recently discovered rhodopsin ion pump (DeNaR, also known as KR2) in the marine bacterium Dokdonia eikasta uses light to pump protons or sodium ions from the cell depending on the ionic composition of the medium. In cells suspended in a KCl solution, DeNaR functions as a light-driven proton pump, whereas in a NaCl solution, DeNaR conducts light-driven sodium ion pumping, a novel activity within the rhodopsin family. These two distinct functions raise the questions of whether the conformations of the protein differ in the presence of K(+) or Na(+) and whether the helical movements that result in the canonical E → C conformational change in other microbial rhodopsins are conserved in DeNaR.

Spectroscopic evidence of the role of an axial ligand histidinate in the mechanism of adrenal cytochrome b561.

Adrenal cytochrome b(561) (AdCytb) is the prototype of a widespread protein family that specializes in delivering electrons donated by ascorbic acid for different processes in eukaryotic cells. AdCytb transports redox equivalents from cytoplasmic ascorbate across the membranes of chromaffin granules to support norepinephrine synthesis within their matrix. The interaction of AdCytb with ascorbate is central to a proposed mechanism of AdCytb's function, and a histidine in the active site of AdCytb was suggested to bind cytoplasmic ascorbate and serve as the acceptor of the proton released during ascorbate oxidation.

Functional and structural roles of residues in the third extramembrane segment of adrenal cytochrome b561.

Several residues in the third extramembrane segment (EM3) of adrenal cytochrome b(561) have been proposed to be involved in this cytochrome's interaction with ascorbate, but there has been no systematic evaluation of residues in the segment. We used alanine scanning mutagenesis to assess the functional and structural roles of the EM3 residues and several adjacent residues (residues 70-85) in the bovine cytochrome. Each alanine mutant was expressed in a bacterial system, solubilized with detergent, and affinity-purified.

1H NMR, mechanism, and mononuclear oxidative activity of the antibiotic metallopeptide bacitracin: the role of D-Glu-4, interaction with pyrophosphate moiety, DNA binding and cleavage, and bioactivity.

The peptidyl antibiotic bacitracin (Bc) is one of the most widely used antibiotics which can bind divalent transition metal ions, including Mn(II), Co(II), Ni(II), Cu(II), and Zn(II). The metal binding is essential for its antimicrobial activity. Previous analysis of the hyperfine-shifted (1)H NMR signals of Co(II)-Bc A(1) revealed the structure of the metal binding environment and a potential hydrophobic site important for the bioactivity of this antibiotic.

Methionine does not reduce Cu(II)-beta-amyloid!--rectification of the roles of methionine-35 and reducing agents in metal-centered oxidation chemistry of Cu(II)-beta-amyloid.

The potential risk of metal-centered oxidative catalysis has been overlooked in the research of the copper complexes of the Alzheimer's disease-related beta-amyloid (Abeta) peptides. Cu(2+) complexes of Abeta(1-40) and its 1-16 and 1-20 fragments have recently been shown to exhibit significant metal-centered oxidative activities toward several catecholamine neurotransmitters with and without H(2)O(2) around neutral pH [G.F.

His92 and His110 selectively affect different heme centers of adrenal cytochrome b(561).

Adrenal cytochrome b(561) (cyt b(561)), a transmembrane protein that shuttles reducing equivalents derived from ascorbate, has two heme centers with distinct spectroscopic signals and reactivity towards ascorbate. The His54/His122 and His88/His161 pairs furnish axial ligands for the hemes, but additional amino acid residues contributing to the heme centers have not been identified. A computational model of human cyt b(561) (Bashtovyy, D.

Overexpression and mechanistic characterization of blastula protease 10, a metalloprotease involved in sea urchin embryogenesis and development.

Blastula protease 10 (BP10) is a metalloenzyme involved in sea urchin embryogenesis, which has been assigned to the astacin family of zinc-dependent endopeptidases. It shows greatest homology with the mammalian tolloid-like genes and contains conserved structural motifs consistent with astacin, tolloid, and bone morphogenetic protein 1. Astacin, a crustacean digestive enzyme, has been proposed to carry out hydrolysis via a metal-centered mechanism that involves a metal-coordinated "tyrosine switch.

Catechol oxidase activity of di-Cu2+-substituted aminopeptidase from Streptomyces griseus.

Streptomyces griseus aminopeptidase exhibits activities toward the hydrolyses of peptides and bis(p-nitrophenyl)phosphate (40 billion fold) and catechol oxidation reported herein with catalytic efficiency (kcat/Km) only about 10 times smaller than that of gypsywort catechol oxidase. The multifunctionality of this enzyme suggests that it is a unique system for further exploration of protein structure and function and a template for design of enzymes of diverse activities.

Catechol oxidase-like oxidation chemistry of the 1-20 and 1-16 fragments of Alzheimer's disease-related beta-amyloid peptide: their structure-activity correlation and the fate of hydrogen peroxide.

The Cu2+ complexes of the 1-16 and the 1-20 fragments of the Alzheimer's disease-related beta-amyloid peptide (CuAbeta) show significant oxidative activities toward a catechol-like substrate trihydroxylbenzene and plasmid DNA cleavage. The latter reflects possible oxidative stress to biological macromolecules, yielding supporting data to the pathological role of these soluble Abeta fragments. The former exhibits enzyme-like kinetics and is dependent on [H2O2], exhibiting k(cat) of 0.