Patrick M. Kelley

The role of chloride ion in Photosystem II I. Effects of chloride ion on Photosystem II electron transport and on hydroxylamine inhibition

1. Chloroplasts washed with Cl--free, low-salt media (pH 8) containing EDTA, show virtually no DCMU-insensitive silicomolybdate reduction. The activity is readily restored when 10 mM Cl- is added to the reaction mixture. Very similar results were obtained with the other Photosystem II electron acceptor 2,5-dimethylquinone (with dibromothymoquinone), with the Photosystem I electron acceptor FMN, and also with ferricyanide which accepts electrons from both photosystems. 2. Strong Cl--dependence of Hill activity was observed invariably at all pH values tested (5.5--8.3) and in chloroplasts from three different plants: spinach, tobacco and corn (mesophyll). 3. In the absence of added Cl- the functionally Cl--depleted chloroplasts are able to oxidize, through Photosystem II, artificial reductants such as catechol, diphenylcarbazide, ascorbate and H2O2 at rates which are 4--12 times faster than the rate of the residual Hill reaction. 4. The Cl--concentration dependence of Hill activity with dimethylquinone as an electron acceptor is kinetically consistent with the typical enzyme activation mechanism: E(inactive) + Cl- in equilibrium E . Cl- (active), and the apparent activation constant (0.9 mM at pH 7.2) is unchanged by chloroplast fragmentation. 5. The initial phase of the development of inhibition of water oxidation in Cl--depleted chloroplasts during the dark incubation with NH2OH (1/2 H2SO4) is 5 times slower when the incubation medium contains Cl- than when the medium contains NH2OH alone or NH2OH plus acetate ion. (Acetate is shown to be ineffective in stimulating O2 evolution).

Vitamins C and E donate single hydrogen atoms in vivo

The antioxidant vitamins, C and E, eliminate cytotoxic free radicals by redox cycling. Energetic and kinetic considerations suggest that cycling of vitamin C and vitamin E between their reduced and free radical forms occurs via the transfer of single hydrogen atoms rather than via separate electron transfer and protonation reactions. This may enable these vitamins to reduce many of the damaging free radicals commonly encountered by biological systems while minimizing the reduction of molecular oxygen to superoxide.

Mechanism of Ascorbic Acid Regeneration Mediated by Cytochrome b561

In summary, ascorbic acid serves as a one-electron donor for dopamine beta-hydroxylase in chromaffin vesicles and probably for peptide amidating monooxygenase in neurohypophyseal secretory vesicles. It appears that the semidehydroascorbate that is produced is reduced by cytochrome b561 to regenerate intravesicular ascorbate. Cytochrome b561, a transmembrane protein, is reduced in turn by an extravesicular electron donor, probably cytosolic ascorbic acid. It will be interesting to see whether other ascorbate-requiring enzymes in other organelles use a similar ascorbate-regenerating system to provide an intravesicular supply of reducing equivalents.

Ketene photochemistry. Relative CH2(1A1) quantum yields at 3130, 3340 and 3660 Å

Abstract The photochemistry of ketene has been studied at 3130, 3340 and 3660 A. A chemical trapping technique was used to determine the relative singlet methylene quantum yields. At a pressure of ≈ 120 torr the 3130:3340:3660 A CH 2 ( 1 A 1 ) quantum yield ratio is 1.0:0.16:

A bimolecular mechanism for ketene photodissociation in the near ultraviolet

The photochemistry of ketene has been studied at 3130, 3340, and 3660 A. Quantum yields for carbon monoxide, ethylene, ethane, and acetylene were measured versus pressure. As p→0 the carbon monoxide quantum yield approaches 2 at each of the three wavelengths. Ethane and acetylene exhibit linear Stern–Volmer plots at 3130 and 3340 A. However, at 3660 A no acetylene was detected and ethane is formed in trace amounts. The above measurements are interpreted in terms of a mechanism which involves adduct formation between the 1A1, and 1A2(1A″) electronic states of ketene.

Concerted proton-electron transfer between ascorbic acid and cytochrome b561.

Ascorbic acid is an essential reductant in biology but its reducing power is paradoxical. At physiological pH the predominant form of ascorbate (the monoanion) is a poor electron donor because it oxidizes to the energetically unfavorable neutral free radical. The ascorbate dianion forms the relatively stable semidehydroascorbate radical anion and is a powerful electron donor but its concentration at neutral pH is insufficient to produce the reaction rates observed. For example, ascorbate rapidly reduces cytochrome b561 from adrenal medullary chromaffin vesicles. This fast reaction rate may be rationalized by a mechanism involving concerted proton-electron transfer rather than electron transfer alone. This would permit reduction of the cytochrome by the abundant ascorbate monoanion but would circumvent formation of unfavorable intermediates. This may be a general mechanism of biological ascorbic acid utilization: enzymes using ascorbic acid may react with the ascorbate monoanion via concerted proton-electron transfer.

Cytochrome b561 and the Maintenance of Redox Poise in Secretory Vesicles

The membranes of a variety of secretory vesicles contain an electron transfer system which supports redox reactions catalyzed by enzymes contained within the vesicles. These enzymes include two important monooxygenases, dopamine s-hydroxylase and peptide amidating monooxygenase. Dopamine shydroxylase, responsible for the biosynthesis of norepinephrine and epinephrine, is found in catecholamine-storing vesicles such as the chromaffin vesicles of the adrenal medulla. Peptide amidating monooxygenase, responsible for the α-amidation of peptide hormones, is found in peptide-storing vesicles such as those in the pituitary and gut. Both monooxygenases use ascorbic acid to reduce the second atom of molecular oxygen to water. While oxygen will diffuse across the secretory vesicle membrane, ascorbate will not. It is to maintain intravesicular ascorbate that the secretory-vesicle membrane has an electron transfer system.

Kinetics of Ferricyanide Reduction by Ascorbate‐loaded Chromaffin‐Vesicle Ghosts

Ascorbate-loaded chromaffin-vesicle ghosts will reduce external ferricyanide.’.’ This ability to transfer electrons across the vesicle membrane is attributed to the membrane protein cytochrome b,,,U and is thought to be used in vivo to take up electrons for the regeneration of intravesicular ascorbic acid (Njus et al.,’ this volume). The reduction of ferricyanide by ascorbate-loaded ghosts is shown in FIGURE 1B.1 Upon addition of ferricyanide, there is a rapid and nearly complete oxidation of cytochrome b,,, (FIG. 1A). The cytochrome stays oxidized until almost no femcyanide remains and then returns to its reduced state. When excess ferricyanide is added, the cytochrome becomes oxidized and remains so, since the internal supply of ascorbate is exhausted. This demonstrates that cytochrome b,,, can accept electrons from ascorbic acid internally, transfer the electrons across the membrane, and reduce ferricyanide on the outside. This shows that cytochrome b561 can transfer electrons across the chromaffin-vesicle membrane, although it does not prove that the cytochrome is the only transmembrane electron carrier. That cytochrome b,,, is at least the principal transmembrane electron carrier is suggested by kinetic arguments. By recording rates of steady-state electron transfer from internal ascorbate to external femcyanide, we can determine rate constants for the transfer of electrons from ascorbate to the membrane (k,,) and for the transfer of electrons from the membrane to femcyanide (k,) (see Njus et al.,’ this volume). These rate constants are determined solely from measurements of the total rate of ferricyanide reduction and thus apply to total electron transfer across the chromaEn-vesicle membrane. If most or all of these electrons are being carried by cytochrome bs61, then the rate constants should also describe the kinetics of cytochrome b,,, oxidation and reduction. To test this, we have performed a computer simulation of the experiment shown in FIGURE 1. We assume that electron transfer occurs through four simple bimolecular reactions b: