The interaction of chloride, fluoride and phosphate ions with the molybdenum centre of sulphite oxidase in the pH range 6.2 to 9.6 has been studied by e.p.r. of Mo(V) in the enzyme reduced by sulphite. Detailed studies were made from e.p.r. spectra recorded at about 120K and more limited studies from spectra of liquid samples at about 295K and also from enzyme activity measurements. Interconversion between low-pH and high-pH Mo(V) e.p.r. signal-giving species [described by Lamy, Gutteridge & Bray (1980) Biochem. J. 185, 397-403] is influenced by chloride concentration, a 10-fold increase in concentration (in the range of about 1 mM to 100 mM) causing an increase of about 1 pH unit in the apparent pK for the conversion. This suggests that chloride is a constituent of the low-pH species. In support of this, high concentrations of fluoride modified the e.p.r. spectrum. Partial conversion to a Mo(V) species, in which F- has presumably replaced Cl- and showing hyperfine coupling of A(19F)av. 0.5mT, is indicated. It is proposed that interconversion between high-pH and low-pH species is of the form: (formula; see text) No evidence that Cl- is essential for enzymic activity was found. Data relating to equilibria amongst low-pH, high-pH and also the phosphate species are presented. Depending on pH and on concentrations of Cl- and H2PO4-, one, two, or all three species may be present. Qualitatively, under appropriate conditions, the phosphate species tends to replace some or all of the low-pH species. Quantitative analysis by a computer procedure permitted an appropriate scheme to be deduced and equilibrium constants to be evaluated. Studies on the e.p.r. signals at 295K indicated that similar equilibria applied in liquid solution, but with some changes in the values of the constants. The structure of the molybdenum centre in its various states and the nature of the enzymic reaction are discussed.

Reduction of sulphite oxidase by sulphite at low pH values in Mes (4-morpholine-ethanesulphonic acid) buffer gives rise to a new molybdenum(V) electron-paramagnetic-resonance spectrum different from that obtained by photoreduction of the enzyme in the same medium. The spectrum is attributed to a sulphite complex of the enzyme, showing g-values of about 2.000, 1.972 and 1.963. The complex is analogous to that with the inhibitor phosphate in that it gives rise to no observable hyperfine coupling of Mo(V) to exchangeable protons.

Using a placebo-controlled, double-blinded, within-participants, randomized, cross-over design, we examined the neurocognitive effects of a: (a) caffeine-containing, adaptogenic herbal-rich natural energy shot (e+ shot), (b) a matched caffeine-containing shot (caffeine), and, (c) a placebo. Participants (n = 30) were low consumers of caffeine without elevated feelings of energy. Before and three times after beverage consumption, a 27-min battery was used to assess motivation to perform cognitive tasks, mood, attention ((serial subtractions of 3 (SS3) and 7 (SS7), the continuous performance task (CPT), and the rapid visual input processing tasks)), heart rate (HR), blood pressure (BP), and motor coordination (nine-hole peg test) with a 10-min break between each post-consumption battery. The procedure was repeated for each beverage for each participant at least 48 h apart and within 30 min the same time of day using a random group assignment with blinding of researchers and subjects. To evaluate for changes in outcomes, a Treatment × Time analysis of covariance controlling for hours of prior night’s sleep was used. Analysis of all outcomes and all treatment comparisons indicated that compared to placebo, both e+ shot ( Δ ¯   = 2.60; η2 = 0.098) and caffeine ( Δ ¯   = 5.30, η2 = 0.098) increased systolic BP 30 min post consumption (still within normal healthy ranges). The caffeine beverage also led to an improvement in most cognitive measures and moods 30-min post-consumption with improvements tapering at 69 and 108 min, while e+ shot noted more steady improvements with no significant differences between beverages on most cognitive and mood measures at 69 and 108 min. However, compared to caffeine, e+shots noted a significant decrease in reaction time at 108 min, while caffeine noted a small change in the opposite direction. No side-effects were reported by any intervention. These results suggest that the specific blend of adaptogens in e+ shot may modulate the neurocognitive effects of caffeine on mood, and cognition.

Background Ferritins are ubiquitous multi-subunit iron storage and detoxification proteins that play a critical role in iron homeostasis. Ferrous ions that enter the protein's shell through hydrophilic channels are rapidly oxidized at dinuclear centers on the H-subunit before transfer to the protein's cavity for storage. The mechanisms of iron loading have been extensively studied but little is known about iron mobilization. Fe(III) reduction can occur via rapid reduction by suitable reducing agents followed by chelation of Fe(II) ions or via direct and slow Fe(III) chelation. Here, the iron release kinetics from ferritin by FMNH2 in the presence of various chaotropic agents are studied and their in-vivo physiological significance discussed. Methods The iron release kinetics from horse and human ferritins by FMNH2 were monitored at 522 nm where the Fe(II)–bipyridine complex absorbs. The experiments were performed in the presence of different concentrations of three chaotropic agents, urea, guanidine HCl, and triton. Results and Conclusions Under our experimental conditions, iron reductive mobilization by the non-enzymatic FMN/NAD(P)H system is limited by the concentration of FMNH2 and is independent on the type or amount of chaotropes present. Diffusion of FMNH2 through the ferritin pores is an unlikely mechanism for ferritin iron reduction. An iron mobilization mechanism involving rapid electron transfer through the protein shell is discussed. General significance Caution must be exercised when interpreting the kinetics of iron mobilization from ferritin using the FMN/NAD(P)H system. The kinetics are highly dependent on the amount of dissolved oxygen and the concentration of reagents used.

Iron uptake into mammalian ferritins reveals oxygen (but not iron) saturation kinetics and physiologically relevant Km,O2 values.