PubMed · 6959907
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C E Graves, P G Fotos. 1982. Ascorbic acid nutritional screening.. https://pubmed.ncbi.nlm.nih.gov/6959907/
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The interaction of Fe(II) and Fe(III) with the 'high-affinity Mn-binding site' in Mn-depleted photosystem II (PSII) was investigated using diphenilcarbazide (DPC)/2,6-dichlorophenol-indophenol (DCIP) inhibition assay. Fe(III) was ineffective in the inhibition of DPC-DCIP reaction while Fe(II) decreased the rate of DCIP photoreduction supported by DPC in the same concentration range as Mn(II). The effectivity of the interaction of Fe(II) with the high affinity Mn-binding site depends on different anions in the same manner as for Mn(II) and coincides with hierarchy observed for the stimulation of O2 evolution. The Fe(II) binding is accompanied by its oxidation. By using reductants it was shown that the high affinity site contains a redox-active component and the reduction of this component totally prevents the binding of Fe(II).
The enzyme kinetics of the FAD-containing membrane-associated D-lactate dehydrogenase (D-LDH) of Escherichia coli have been investigated by stopped-flow spectroscopy. The reduction of D-LDH by the substrate, D-lactate, exhibits a two-stage behavior as observed by the absorbance change for the enzyme-bound FAD. The fast stage with a maximum rate of 400 s-1 represents the rapid formation of the enzyme-substrate complex and the formation of the equilibrium between the oxidized and the reduced enzyme-substrate complexes. The slow stage, which occurs on the order of 0.36 s-1, represents the slow release of the product, pyruvate, from the reduced enzyme. The formation of a D-LDH semiquinone radical was not observed during the oxidation of D-lactate by D-LDH at 25 degrees C. However, during the subsequent electron transfer from the reduced enzyme to a nitroxide spin-label, a one-electron acceptor, an enzyme intermediate has been observed and identified by both optical and EPR spectroscopies as an anionic semiquinone. Results from 1H-NMR spectroscopic studies suggest the possible formation of a substrate carbanion when D-lactate is bound at the active site of D-LDH.
The requirement for Ca2+ in the Mn(2+)-dependent photoactivation of oxygen evolution was re-evaluated using 17 kDa/24 kDa-less photosystem II (PSII) membranes depleted of (Mn)4-clusters by NH2OH extraction. At optimum conditions (1 mM Mn2+/10 microM 2,6-dichlorphenolindophenol (DCIP)/20 mM Ca2+), the light-induced increase of oxygen-evolution activity, the increase of membrane-bound Mn, and the B-band thermoluminescence emission intensity occurred in parallel. The extent of recovery of the oxygen-evolution activity was equivalent to 88% and 66% of the activity shown by parent NaCl-extracted PSII membranes and by PSII membranes, respectively. Neither photodamage of primary electron transport nor photoligation of nonfunctional Mn > or = 3+ occurred. Analyses of the Ca2+ concentration dependence for the maximum recovery of oxygen evolution activity gave evidence for Ca(2+)-binding site(s) having Km values of approximately 38 and approximately 1300 microM. Illumination of membranes in the strict absence of Ca2+ resulted in large increases (up to 18 Mn/200 chlorophyll) of EDTA nonextractable, EPR silent, nonfunctional membrane-bound Mn > or = 3+ and small increases of oxygen-evolution capability, dependent on pH and concentrations of Mn2+ and DCIP. No photodamage of primary electron transport and only approximately 17% decrease of AT-band thermoluminescence occurred during the photoligation of the Mn > or = 3%. In the strict absence of Ca2+, significant recovery of oxygen-evolution activity was obtained under a limited set of conditions permitting photoligation of a limited abundance of the nonfunctional Mn > or = 3+. Small (NH2-OH, H2O2) as well as bulky external reductants readily reduced and dissociated the Mn > or = 3+ from the membranes. Reillumination of these membranes under optimal conditions for photoactivation (plus Ca2+) gave a high yield of (Mn)4-clusters and oxygen-evolution capability. Similarly, simple addition of Ca2+ to membranes containing nonfunctional Mn > or = 3+ followed by reillumination resulted in the conversion of Mn > or = 3+ to (Mn)4-clusters. It is argued that Ca2+ promotes the conformational change involved in the conversion of the Mn2+ mononuclear intermediate to the Mn(3+)-Mn2+ binuclear intermediate in the photoactivation mechanism, thereby permitting photoassembly of (Mn)4-clusters and preventing photo-inactivation by Mn > or = 3+ ions.