Large anions induce H-production from the nitrogenase MoFe proteins of Clostridium Pasteurianum and Azotobacter vinelandii.

In the absence of sodium dithionite (DT), addition of the large anions Br, I and HS to the MoFe proteins of Azotobacter vinelandii (Av1) and Clostridium pasteurianum (Cp1) released ~1.0 H/MoFe protein with an associated increase in the absorbance from 400 to 800 nm, indicative of protein oxidation. The reaction of I with Cp1 released ~1.

Nucleotide-assisted [Fe4S4] redox state interconversions of the Azotobacter vinelandii Fe protein and their relevance to nitrogenase catalysis.

The [Fe4S4] cluster of the nitrogenase Fe protein from Azotobacter vinelandii can exist in three redox states: oxidized [Fe4S4](2+), dithionite reduced [Fe4S4](1+), and two forms of the all ferrous [Fe4S4](0), S = 4 and 0. Operation of the [Fe4S4](2+)/ [Fe4S4](1+) redox couple transfers one electron to the MoFe protein during catalysis with hydrolysis of two MgATPs. In contrast, the [Fe4S4](2+)/[Fe4S4](0) couple transfers two electrons per binding event, accompanied by hydrolysis of only two MgATPs.

Anion deposition into ferritin.

When the iron core of equine spleen ferritin is reduced, anions in solution cross the protein shell and enter the ferritin interior as part of a charge balancing reaction. Anion sequestration inside ferritin during iron core reduction was monitored using ion selective electrodes, inductively coupled plasma emission, and energy-dispersive X-ray spectroscopy. The requirement for anion translocation to the ferritin interior occurs because upon iron core reduction, two OH(-) ions per iron are released or neutralized inside ferritin leaving a net positive charge.

Non-reductive iron release from horse spleen ferritin using desferoxamine chelation.

The rate of Fe(3+) release from horse spleen ferritin (HoSF) was measured using the Fe(3+)-specific chelator desferoxamine (DES). The reaction consists of two kinetic phases. The first is a rapid non-linear reaction followed by a slower linear reaction.

Gold nanoshell assembly on a ferritin protein employed as a bio-template.

Gold nanoshells around 26 nm in diameter with a 7 nm thick wall were fabricated in an aqueous solution using pre-reconstituted ferritin proteins as a removable bio-template. The synthesis of gold nanoshells was initiated by planting gold nanoparticle seeds in the hydrophilic three-fold channels of the ferritin protein. The process was facilitated by the energetically favorable gold-sulfur bonds formed at the cysteine residues lining these channels.

Anaerobic iron deposition into horse spleen, recombinant human heavy and light and bacteria ferritins by large oxidants.

Large-molecule oxidants oxidize Fe(II) to form Fe(III) cores in the interior of ferritins at rates comparable to or faster than the iron deposition reaction using O(2) as oxidant. Iron deposition into horse spleen ferritin (HoSF) occurs using ferricyanide ion, 2,6-dichlorophenol-indophenol, and several redox proteins: cytochrome c, stellacyanin, and ceruloplasmin. Cytochrome c also loads iron into recombinant human H-chain (rHF), human L-chain (rLF), and A.

Flavodoxin hydroquinone reduces Azotobacter vinelandii Fe protein to the all-ferrous redox state with a S = 0 spin state.

Azotobacter vinelandii flavodoxin hydroquinone (FldHQ) is a physiological reductant to nitrogenase supporting catalysis that is twice as energy efficient (ATP/2e- = 2) as dithionite (ATP/2e- = 4). This catalytic efficiency results from reduction of Fe protein from A. vinelandii (Av2) to the all-ferrous oxidation state ([Fe4S4]0), in contrast to dithionite, which only reduces Av2 to the [Fe4S4]1+ state.

Ferritin-catalyzed consumption of hydrogen peroxide by amine buffers causes the variable Fe2+ to O2 stoichiometry of iron deposition in horse spleen ferritin.

Ferritin catalyzes the oxidation of Fe2+ by O2 to form a reconstituted Fe3+ oxy-hydroxide mineral core, but extensive studies have shown that the Fe2+ to O2 stoichiometry changes with experimental conditions. At Fe2+ to horse spleen ferritin (HoSF) ratios greater than 200, an upper limit of Fe2+ to O2 of 4 is typically measured, indicating O2 is reduced to 2H2O. In contrast, a lower limit of Fe2+ to O2 of approximately 2 is measured at low Fe2+ to HoSF ratios, implicating H2O2 as a product of Fe2+ deposition.

Electron exchange between Fe(II)-horse spleen ferritin and Co(III)/Mn(III) reconstituted horse spleen and Azotobacter vinelandii ferritins.

Azotobacter vinelandii bacterioferritin (AvBF) containing 800-1500 Co or Mn atoms as Co(III) and Mn(III) oxyhydroxide cores (Co-AvBF, Mn-AvBF) was synthesized by the same procedure used previously for horse spleen ferritin (HoSF). The kinetics of reduction of Co-AvBF and Mn-AvBF by ascorbic acid are first-order in each reactant. The rate constant for the reduction of Mn-AvBF (8.

Evidence for a synergistic salt-protein interaction -- complex patterns of activation vs. inhibition of nitrogenase by salt.

The molybdenum nitrogenase enzyme system, comprised of the MoFe protein and the Fe protein, catalyzes the reduction of atmospheric N(2) to NH(3). Interactions between these two proteins and between Fe protein and nucleotides (MgADP and MgATP) are crucial to catalysis. It is well established that salts are inhibitors of nitrogenase catalysis that target these interactions.

Rate of iron transfer through the horse spleen ferritin shell determined by the rate of formation of Prussian Blue and Fe-desferrioxamine within the ferritin cavity.

Iron (2+ and 3+) is believed to transfer through the three-fold channels in the ferritin shell during iron deposition and release in animal ferritins. However, the rate of iron transit in and out through these channels has not been reported. The recent synthesis of [Fe(CN)6]3-, Prussian Blue (PB) and desferrioxamine (DES) all trapped within the horse spleen ferritin (HoSF) interior makes these measurements feasible.

Cobalt oxide hollow nanoparticles derived by bio-templating.

We present here the first fabrication of hollow cobalt oxide nanoparticles produced by a protein-regulated site-specific reconstitution process in aqueous solution and describe the metal growth mechanism in the ferritin interior.

Kinetic and thermodynamic characterization of the cobalt and manganese oxyhydroxide cores formed in horse spleen ferritin.

Horse spleen ferritin (HoSF) containing 800-1500 cobalt or 250-1200 manganese atoms as Co(O)OH and Mn(O)OH mineral cores within the HoSF interior (Co-HoSF and Mn-HoSF) was synthesized, and the chemical reactivity, kinetics of reduction, and the reduction potentials were measured. Microcoulometric and chemical reduction of HoSF containing the M(O)OH mineral core (M = Co or Mn) was rapid and quantitative with a reduction stoichiometry of 1.05 +/- 0.

Kinetic studies of iron deposition catalyzed by recombinant human liver heavy and light ferritins and Azotobacter vinelandii bacterioferritin using O2 and H2O2 as oxidants.

The discrepancy between predicted and measured H(2)O(2) formation during iron deposition with recombinant heavy human liver ferritin (rHF) was attributed to reaction with the iron protein complex [Biochemistry 40 (2001) 10832-10838]. This proposal was examined by stopped-flow kinetic studies and analysis for H(2)O(2) production using (1) rHF, and Azotobacter vinelandii bacterial ferritin (AvBF), each containing 24 identical subunits with ferroxidase centers; (2) site-altered rHF mutants with functional and dysfunctional ferroxidase centers; and (3) recombinant human liver light ferritin (rLF), containing no ferroxidase center. For rHF, nearly identical pseudo-first-order rate constants of 0.

Electrical conductivity of ferritin proteins by conductive AFM.

Electrical conductivity measurements were performed on single apoferritin and holoferritin molecules by conductive atomic force microscopy. Conductivity of self-assembled monolayer films of ferritin molecules on gold surfaces was also measured. Holoferritin was 5-15 times more conductive than apoferritin, indicating that for holoferritin most electron-transfer goes through the ferrihydrite core.

Kinetic studies of iron deposition in horse spleen ferritin using H2O2 and O2 as oxidants.

The reaction of horse spleen ferritin (HoSF) with Fe2+ at pH 6.5 and 7.5 using O2, H2O2 and 1:1 a mixture of both showed that the iron deposition reaction using H2O2 is approximately 20- to 50-fold faster than the reaction with O2 alone.

Kinetic studies of iron deposition in horse spleen ferritin using O2 as oxidant.

An optical flow cell provided a means to conveniently measure the rate of successive Fe(2+) oxidation reactions catalyzed by horse spleen ferritin (HoSF) to determine if both ferroxidase and mineral core Fe(2+) oxidation reactions occur. The oxygen concentration and pH were held constant and multiple additions of Fe(2+)/HoSF ratios of 1, 10, 100, 150, 250 and 400 were conducted, creating core sizes ranging from 12 to 2800. During these oxidations, the absence of nonspecific Fe(OH)(3) formation and the presence (>95%) of Fe(OH)(3) deposited within the core of HoSF demonstrated the validity of monitoring iron deposition into HoSF by this procedure.