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The objective of this study was to clarify the mechanism of electron transfer in the human neuronal nitric oxide synthase (nNOS) flavin domain using the recombinant human nNOS flavin domains, the FAD/NADPH domain (contains FAD- and NADPH-binding sites), and the FAD/FMN domain (the flavin domain including a calmodulin-binding site). The reduction by NADPH of the two domains was studied by rapid-mixing, stopped-flow spectroscopy. For the FAD/NADPH domain, the results indicate that FAD is reduced by NADPH to generate the two-electron-reduced form (FADH(2)) and the reoxidation of the reduced FAD proceeds via a neutral (blue) semiquinone with molecular oxygen or ferricyanide, indicating that the reduced FAD is oxidized in two successive one-electron steps. The neutral (blue) semiquinone form, as an intermediate in the air-oxidation, was unstable in the presence of O(2). The purified FAD/NADPH domain prepared under our experimental conditions was activated by NADP(+) but not NAD(+). These results indicate that this domain exists in two states; an active state and a resting state, and the enzyme in the resting state can be activated by NADP(+). For the FAD/FMN domain, the reduction of the FAD-FMN pair of the oxidized enzyme with NADPH proceeded by both one-electron equivalent and two-electron equivalent mechanisms. The formation of semiquinones from the FAD-FMN pair was greatly increased in the presence of Ca(2+)/CaM. The air-stable semiquinone form, FAD-FMNH(.), was further rapidly reduced by NADPH with an increase at 520 nm, which is a characteristic peak of the FAD semiquinone. Results presented here indicate that intramolecular one-electron transfer from FAD to FMN is activated by the binding of Ca(2+)/CaM.

The conditions for the heterologous expression of both untagged and His-tagged human sulfite oxidase in Escherichia coli have been optimized. Maximum production of active enzyme requires expression in a mob- cell strain at low levels of the inducer. Using these conditions, 3.9-5.6 mg of untagged and 15 mg of His-tagged sulfite oxidase were isolated per liter of cell culture. These represent significantly higher levels than previously reported for any molybdopterin-containing protein. High levels of enzyme activity and molybdenum incorporation were maintained despite the increase in yield, and no significant differences in kinetic properties were observed between the tagged and untagged sulfite oxidase. Additionally, the molybdenum domain of sulfite oxidase was expressed in a stable, active form as a His-tagged protein. The molybdenum domain was also expressed in the presence of tungstate to enable examination of the molybdopterin-tungsten form of sulfite oxidase.

A synthetic flavocytochrome with the reductase and oxygenase activities was obtained by covalent binding of riboflavin to cytochrome P450 2B4. The reactions catalyzed by the newly synthesized flavocytochromes were studied. Formation of carbon monoxide complex with the reduced form of hemoprotein led to 60–80% inhibition of oxygenase reactions, indicating the leading role of reduced heme iron in generating active oxygen species by flavocytochromes.

Ascorbic acid can recycle alpha-tocopherol from the tocopheroxyl free radical in lipid bilayers and in micelles, but such recycling has not been demonstrated to occur across cell membranes. In this work the ability of intracellular ascorbate to protect and to recycle alpha-tocopherol in intact human erythrocytes and erythrocyte ghosts was investigated. In erythrocytes that were 80% depleted of intracellular ascorbate by treatment with the nitroxide Tempol, both 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) and ferricyanide oxidized alpha-tocopherol to a greater extent than in cells not depleted of ascorbate. In contrast, in erythrocytes in which the intracellular ascorbate concentration had been increased by loading with dehydroascorbate, loss of alpha-tocopherol was less with both oxidants than in control cells. Protection against AAPH-induced oxidation of alpha-tocopherol was not prevented by extracellular ascorbate oxidase, indicating that the protection was due to intracellular and not to extracellular ascorbate. Incubation of erythrocytes with lecithin liposomes also generated an oxidant stress, which caused lipid peroxidation in the liposomes and depleted erythrocyte alpha-tocopherol, leading to hemolysis. Ascorbate loading of the erythrocytes delayed liposome oxidation and decreased loss of alpha-tocopherol from both cells and from alpha-tocopherol-loaded liposomes. When erythrocyte ghosts were resealed to contain ascorbate and challenged with free radicals generated by AAPH outside the ghosts, intravesicular ascorbate was totally depleted over 1 h of incubation, whereas alpha-tocopherol decreased only after ascorbate was substantially oxidized. These results suggest that ascorbate within the erythrocyte protects alpha-tocopherol in the cell membrane by a direct recycling mechanism.

Incubation of either Chlorella nitrate reductase or the recombinant flavin domain of spinach nitrate reductase with reagents specific for modification of cysteine residues, such as N-ethylmaleimide, resulted in a time-dependent inactivation of NADH:ferricyanide reductase activity which could be prevented by incubation in the presence of NADH. At 25 degrees C and employing a fixed enzyme:modifier ratio, the rate of inactivation for both the Chlorella and spinach enzymes followed the order p-chloromercuribenzoate > methyl methanethiosulfonate > 2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid > N-ethylmaleimide. For the spinach flavin domain, inactivation by methyl methanethiosulfonate or p-chloromercuribenzoate was found to be concentration independent suggesting the absence of nonspecific modifications. Initial rate studies of the methyl methanethiosulfonate-modified flavin domain indicated a reduction in NADH:ferricyanide activity (Vmax) from 85 to 44 micromol NADH consumed/min/nmol FAD and an increase in the Km for NADH from 12 to 35 microM when compared to the native enzyme, confirming a role for cysteine residue(s) in maintaining diaphorase activity. Site-directed mutagenesis of the four individual cysteines (residues 17, 54, 62, and 240) in the recombinant spinach flavin domain resulted in mutant proteins with visible and CD spectra very similar to those of the wild-type domain. Initial rate studies indicated that only substitutions of serine for cysteine 240 decreased diaphorase activity with maximal NADH:ferricyanide activity for the C240S mutant corresponding to 51 micromol NADH consumed/min/nmol FAD with a Km for NADH of 14 microM. Mutation of C240 to Ala or Gly resulted in greater loss of activity. The thermal stability of the four serine mutants was slightly decreased compared to the wild-type domain with the C62S mutant exhibiting the greatest instability. In contrast to the effects on diaphorase activity, square wave voltammetric studies indicated changes in the oxidation-reduction midpoint potential for the FAD/FADH2 couple in the C54S (E0'= -197 mV), C62S (E0' = -226 mV), and C240S (E0' = -219 mV) mutants compared to the wild-type domain (E0' = -268 mV). These results indicate that of the four cysteine residues in the spinach nitrate reductase flavin domain, only C240 plays a role in maintaining diaphorase activity, while C54 has the greatest influence on flavin redox potential and that no correlation between changes in catalytic activity and flavin redox potential was observed.

The reaction mechanism of a 1,4-benzoquinone reductase from the wood-rotting basidiomycete Phanerochaete chrysosporium was investigated. The native, oxidized, FMN-containing enzyme was reduced quantitatively by NADH and the resulting reduced enzyme was reoxidized in the presence of one equivalent of 2,6-di-methoxy-1,4-benzoquinone (DMBQ). The stoichiometry of NADH oxidation versus DMBQ reduction is 1:1. The enzyme catalyzes the reduction of quinones to hydroquinones by a ping-pong steady-state mechanism. However, inhibition is observed at low NADH concentrations. Quinone products derived from the autooxidation of the unstable compounds 1,2,4-trihydroxybenzene and 5-chloro-2,3,4-trihydroxybenzene also appear to be substrates for the quinone reductase. The enzyme reduces the one-electron acceptors ferricyanide and ferricytochrome c (Cc3+) with rates of 58.4 and 0.08%, respectively, compared to DMBQ. The stoichiometry of NADH oxidation versus ferricyanide reduction is 1:2. In the presence of quinones the rates of Cc3+ and ferricyanide reduction are increased, owing to the nonenzymatic reduction of these acceptors by enzyme-generated hydroquinone products. Dicumarol and Cibacron blue are competitive inhibitors with respect to NADH, with Ki values of 2.1 and 0.30 microM, respectively. Reconstitution of the apoprotein with FMN yields a fully active enzyme at an FMN-to-protein ratio of 2:1, suggesting that the flavin content of the enzyme is two molecules of FMN per dimer.

The redox potentials of FAD and FMN of Cytochrome P450 reductase (reductase) are equivalent in solution but differ by 138 mV when bound to reductase. The interaction of each flavin with its flavin binding domain confers the unique electron transferring abilities to each flavin. In order to determine flavin binding properties and activity of the FAD binding domain, we have expressed in pTrcHis three fragments (1161, 1244, and 1556 bp) of rat liver reductase cDNA encompassing the proposed FAD and NADPH binding domain. The FAD binding fragments from cells harboring the 1161- and 1556-bp-containing vectors were stable and bound 0.66 and 0.71 mol FAD/mol enzyme, respectively. Both fragments reduce ferricyanide (54 and 104% of FMN-less reductase/mol bound flavin, respectively) and participate in the transhydrogenation reaction of 3-AcPyADP (41 and 65% of FMN-less reductase/mol bound flavin, respectively). FAD-less fragments were purified and reconstituted with 8-amino-FAD and 8-chloro-FAD to determine binding efficiencies.

The tissue-specific expression and induction of P450 GP-1 , a constitutive form of cytochrome P450 of the guinea pig classified into the CYP2B subfamily, were studied. Prior to these studies, a P450 form (P450 GP-1 PB ) was purified from phenobarbital-treated guinea pigs and the properties were compared with those of the P450 GP-1 . This form was judged to be the same as P450 GP-1 existing in untreated animals by comparisons of their N-terminal amino acid sequences, peptide maps, and affinities toward anti-P450 GP-1 antibody. Immunostaining of P450 GP-1 revealed that the lung and small intestine as well as the liver of untreated guinea pigs contain P450 GP-1 , while none or only small amounts of this P450 form were observed in the kidney, heart, spleen, urinary bladder, and testis. The amount of liver P450 GP-1 protein expressed in untreated guinea pigs was estimated to be 19.4% of the total cytochrome P450 and this form was increased 1.7-fold by phenobarbital treatment. Similarly, intestinal P450 GP-1 was increased by phenobarbital treatment. However, lung P450 GP-1 was not increased by the treatment. It was also observed that the liver P450 GP-1 is induced with SKF-525A to the same extent as with phenobarbital. On the other hand, dexamethasone, p,p′ -dichlorodiphenyltrichloroethane, and isosafrole showed no or only a weak ability to increase the liver P450 GP-1 content. The drug-metabolizing activities in the liver microsomes of SKF-525A-pretreated guinea pigs were lower than those in phenobarbital-treated animals, although the P450 GP-1 protein was induced equally by these treatments. The low activities of SKF-525A-treated animals in the drug metabolisms were attributed to the formation of the metabolic-intermediate complex between P450 GP-1 and SKF-525A metabolite. These results permitted us to conclude that the tissue specificity in the expression of guinea pig P450 belonging to the CYP2B subfamily and the inducibility with chemicals are similar to those of rat CYP2B1, although the constitutive expression of guinea pig liver P450 GP-1 is much higher than that of CYP2B1.

It has been reported that vanadate-stimulated oxidation of NAD(P)H by microsomal systems can proceed anaerobically, in contrast to the general notion that the oxidation proceeds exclusively by an O 2 −1 -dependent free radical chain mechanism. The current study indicates that microsomal systems are endowed with a vanadate-reductase property, involving a NAD(P)H-dependent electron transport cytochrome P450 system. Our ESR measurements demonstrated the formation of a vanadium (IV) species in a mixture containing vanadate, rat liver microsomes, and NAD(P)H. This vanadium(IV) species was identified as the vanadyl ion (VO 2+ ) by comparison with the ESR spectrum of VOSO 4 . The initial rate of vanadium(IV) formation depends linearly on the concentration of microsomes. The Michaelis-Menten constants were found to be: k m = 1.25 mM and V max = 0.066 μ mol (min) −1 (mg microsomes) −1 , respectively. Pretreatment of the microsomes with carbon monoxide or K 3 Fe(CN) 6 reduced vanadium(IV) generation, suggesting that the NAD(P)H-dependent electron transport cytochrome P450 system plays a significant role in the microsomal reduction of vanadate. Measurements under argon or in the presence of superoxide dismutase caused only minor (less than 10%) reductions in vanadium(IV) generation. The VO 2+ species was also detected in NAD(P)H oxidation by fructose plus vanadate, a reaction known to proceed via an O 2 − -mediated chain mechanism. However, the amount of vanadium(IV) generated by this reaction was an order of magnitude smaller than that by the microsomal system and was inhibitable by superoxide dismutase, affirming the conclusion that the microsomal/ NAD(P)H system is endowed with the (O 2 − -independent) vanadium(V) reductase property.

In this work, various ferro-ferricyanide oxidoreductase activities of rat liver mitochondria were studied to find conditions under which the outer membrane might restrict the flux of these highly charged non-biological anions. When the isotonic low ionic strength medium was supplemented with 25 mM KCl, a several-fold increase in the succinate-ferricyanide reductase activity of mitochondria and in the rate of external NADH oxidation in the presence of ferrocyanide was observed. Mitochondrial respiration with 5 mM ferrocyanide was almost completely inhibited after consumption of 3.8–18.5% of the dissolved oxygen, depending on the medium and the presence of 2,4-dinitrophenol. These and other experimental data together with mathematical modeling of the redox-state equilibrium suggest that the measured activities might be restricted by two factors: first, the permeability of the outer mitochondrial membrane and second, a strong influence of the ionic strength of incubation media on the intermembrane space redox reactions.

The gene coding for expression of an endogenous soluble fusion protein comprising a b-type cytochrome-containing domain and a FAD-containing domain has been cloned from rat liver mRNA. The 1461-bp hemoflavoprotein gene corresponded to a protein of 493 residues with the heme- and FAD-containing domains comprising the amino and carboxy termini of the protein, respectively. Sequence analysis indicated the heme and flavin domains were directly analogous to the corresponding domains in microsomal cytochrome b(5) (cb5) and cytochrome b(5) reductase (cb5r), respectively. The full-length fusion protein was purified to homogeneity and demonstrated to contain both heme and FAD prosthetic groups by spectroscopic analyses and MALDI-TOF mass spectrometry. The cb5/cb5r fusion protein was able to utilize both NADPH and NADH as reductants and exhibited both NADPH:ferricyanide (k(cat) = 21.7 s(-1), K(NADPH)(m) = 1 microM. K(FeCN6)(m) = 8 microM) and NADPH:cytochrome c (k(cat) = 8.3 s(-1), K(NADPH)(m) = 1 microM. K(cyt c)(m) = 7 microM) reductase activities with a preference for NADPH as the reduced pyridine nucleotide substrate. NADPH-reduction was stereospecific for transfer of the 4R-proton and involved a hydride transfer mechanism with a kinetic isotope effect of 3.1 for NADPH/NADPD. Site-directed mutagenesis was used to examine the role of two conserved histidine residues, H62 and H85, in the heme domain segment. Substitution of either residue by alanine or methionine resulted in the production of simple flavoproteins that were effectively devoid of both heme and NAD(P)H:cytochrome c reductase activity while retaining NAD(P)H:ferricyanide activity, confirming that the former activity required a functional heme domain. These results have demonstrated that the rat cb5/cb5r fusion protein is homologous to the human variant and has identified the heme and FAD as the sites of interaction with cytochrome c and ferricyanide, respectively. Mutagenesis has confirmed the identity of both axial heme ligands which are equivalent to the corresponding residues in microsomal cytochrome b(5).

Soluble guanylyl cyclase (sGC), an enzyme involved in cGMP signal transduction, is activated by NO binding to the endogenous heme. The mechanism of deactivation is not known. In tissues, cGMP levels decrease within minutes, despite the fact that sGC is activated to levels above the phosphodiesterase activity. Simple dissociation of NO from the heme in sGC has been suggested as a possible deactivation mechanism; however, dissociation rates of NO from ferrous heme proteins are typically very slow. Since oxidants and reductants are known to affect sGC activity, we have tested the effect of a variety of redox-active agents on the activity of NO-activated sGC. All the redox-active compounds tested, covering a wide range of reduction potentials, selectively deactivated the NO-activated sGC while having little or no effect on the basal activity of the enzyme. Among the reagents studied in detail, deactivation of sGC by air occurred slowly, while deactivation by ferricyanide was faster and methylene blue was fastest. The mechanism of deactivation of sGC by dioxygen in the air is straightforward: the heme is oxidized to Fe(III)heme and nitrate is formed. This reaction is similar to that of dioxygen with NOHb and NOMb as occurs in cured meats. Methylene blue and ferricyanide deactivate sGC by a different, as yet undetermined, mechanism.

Quinones which are produced during the mineralization of lignin and xenobiotics by the white rot fungus Phanerochaete chrysosporium were reduced by a plasma membrane redox system of the fungus. Both intracellular enzymes and the plasma membrane redox system were able to reduce 1,l-benzoquinone. However, no quinone reductase activity was observed with the extracellular culture fluid. The intracellular reductase activity had a pH optimum between 6.0 and 7.0 and a K m of 150 μM. Reduction of 1,4-benzoquinone by the plasma membrane redox system had a pH optimum between 7.5 and 8.5 and exhibited saturation kinetics ( K m = 11 μM, V max = 16 nmol/min/mg mycelia dry weight). Ferricyanide totally inhibited the quinone reduction until the ferricyanide was completely reduced by the membrane. Radicals (chlorpromazine and 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS)) that can be generated by the lignin peroxidases were also reduced by the plasma membrane redox system. Reduction of the ABTS cation radical also totally inhibited quinone reduction until the radical was completely reduced. Finally, quinone reduction rates were identical after the reduction of ferricyanide, ABTS cation radical, or quinone, suggesting that the plasma membrane redox system may actually protect the fungus from oxidative damage from free radicals generated by the lignin degrading system.

A transplasma membrane redox system of Phanerochaete chrysosporium was studied using ferricyanide, a membrane-impermeable electron acceptor. Rates of reduction were dependent upon initial ferricyanide concentration and mycelial mass. Specific activities of 12 ± 2 nmol/min/mg mycelia (dry wt) were consistently obtained using nutrient-sufficient mycelia at pH 8.0 and 10 mM ferricyanide. Upon nutrient limitation (either carbon or nitrogen), activity decreased. Reduction was inhibited by carbonyl cyanide m -chloromethoxyphenyl hydrazone, 2,4-dinitrophenol, and sodium azide but not by potassium cyanide at 100 nmol/mg mycelia. Ferricyanide reduction and proton export rates increased with pH above the physiological pH for the fungus. The stimulation in proton exported by the addition of ferricyanide was equal to the rate of ferricyanide reduced at pH 8.0 when Hepes buffer was used. The relevance of these findings with regard to the physiological pH optimum of the fungus and the metabolism of pollutants by this fungus is discussed.

The reaction of phenyldiazene with purified, phenobarbital-inducible rabbit cytochrome P450IIB4, mouse cytochrome P450IIB10, and dog cytochrome P450IIB11 yields complexes with absorbance maxima at 480 nm. Treatment of the cytochrome P450 complexes with K3Fe(CN)6 results in disappearance of the 480-nm absorption. Extraction of the prosthetic group from the proteins after these reactions yields the two isomers of N-phenylprotoporphyrin IX with the N-phenyl group on pyrrole rings A and D as the major products and the regioisomer with the N-phenyl on pyrrole ring C as a minor product. The A:C:D arylated pyrrole ring ratio is 3:2:3 for rabbit P450IIB4, 3:1:3 for mouse P450IIB10, and 4:1:2 for dog P450IIB11. Formation of the A and D regioisomers is consistent with the results obtained previously for rat isozymes IA1, IIB1, IIB2, and IIE1, but the rabbit, mouse, and dog P450IIB enzymes differ from the four rat enzymes in that a substantial amount of the isomer with the N-phenyl on pyrrole ring C is also formed. The results indicate that the region over pyrrole ring B is masked by protein residues in all the active sites and suggest that the region over pyrrole ring C is more hindered by protein residues in the rat than in the rabbit, mouse, or dog enzymes so far examined.

The role of ferritin in catalyzing the oxidation of luminol with the production of chemiluminescence was investigated. The effect of pH was compared to its effect on K3Fe(CN)6-catalyzed oxidation and different pH optima were recorded for the two catalysts. The ferrous iron chelator, bipyridyl, enhanced the production of chemiluminescence catalyzed by FeSO4 and ferritin but had little effect on the K3Fe(CN)6-catalyzed reaction. Desferal reduced the level of chemiluminescence in the presence of FeSO4 and ferritin but was a much more effective inhibitor of chemiluminescence catalyzed by K3Fe(CN)6. The hydroxyl radical scavenger, mannitol, had little effect upon light production whereas superoxide dismutase inhibited light production. The addition of antihuman spleen ferritin completely inhibited activity. The catalytic activity of both H and L rich ferritins was affected by iron content. Activity increased until the Fe/protein ratio reached 0.04 micrograms Fe/micrograms protein and then decreased with increasing iron content. Thus activity is controlled by the iron content of the molecule and influenced by its subunit composition as is the uptake of iron into ferritin. These findings suggest that ferroxidation by ferritin is associated with the ability to generate radicals of the nitrogenous base luminol with the production of chemiluminescence. Although activity is greatest at alkaline pH there is significant activity at pH 7.4. Ferritin therefore may be able to generate free radical reactions in vivo with the acidic isoferritin being most active.

The aerobic metabolism of benzphetamine by liver microsomes, during a cytochrome P-450-catalyzed mixed-function oxidation reaction, results in the formation of an easily detected spectral complex with an absorption band maximum at 456 nm. Electron paramagnetic resonance studies, as well as studies with the chemical reductant, sodium dithionite, or the oxidant, potassium ferricyanide, indicate that the spectral complex results from the formation of a product adduct with reduced cytochrome P-450. The spectral properties of this product complex of cytochrome P-450 have been compared to those observed with carbon monoxide, metyrapone, and ethylisocyanide. The reaction of these reagents to specific pools of microsomal cytochrome P-450 permits the identification of at least two major and two minor types of cytochrome P-450 in liver microsomes prepared from phenobarbital-treated rats.

The reoxidation phase of the catalytic cycle of succinate dehydrogenase was studied in Complex II preparations' by the rapid freeze-electron paramagnetic resonance (epr) technique. With the synthetic water-soluble Q1 analog, 2,3-dimethoxy-5-methyl-6-pentyl-1, 4-benzoquinone (DPB), as the oxidant, the observed reoxidation of the epr-detectable components, previously reduced with dithionite or succinate, came to completion within a few milliseconds, well within the turnover time of the enzyme. Only ~80% of Fe-S center 1 and the HiPIP (the high-potential cluster) Fe-S center reacted rapidly with DPB, however; similarly incomplete reactions were observed previously in our studies of the reduction of the enzyme by succinate. The subsequent addition of ferricyanide, which appears to act as a chemical oxidant in these experiments, caused immediate reoxidation of the Fe-S centers and of the free radical. Ferricyanide and phenazine methosulfate (PMS) reoxidized all epr-detectable components in Complex II as well as in reconstitutively active, soluble preparations in'

Certain mono- and dihydroxybenzene derivatives cause depigmentation of skin and hair, and appear to be selectively cytotoxic for melanized pigment cells. As direct physical and/or chemical interaction between depigmenter (DP) and pigment melanin may play a role in depigmentation, we have carried out preliminary studies in model systems where such interactions may easily be separated from effects due to tyrosinase, melanosomal proteins, and other components. We have used synthetic L-3,4-hydroxyphenylalanine (L-DOPA)-melanin as a protein-free model pigment and potassium ferricyanide as a model electron acceptor. Compounds studied were catechol, 4-t-butylcatechol, 4-methylcatechol, 3,4-dihydroxyphenylalanine (DOPA), 3,4-dihydroxyphenylacetic acid, hydroquinone, 4-methoxyphenol, 4-t-butylphenol, and 2,6, di-t-butyl-4-methylphenol (BHT) in 0.1 M phosphate buffer, pH 7.4. These compounds vary widely in their ability to depigment hair and skin. Ferricyanide reduction by DP in the presence and absence of melanin was monitored spectrophotometrically. The sparingly soluble BHT and 4-t-butylphenol did not reduce ferricyanide in the absence or presence of melanin. For the other compounds, kinetic analysis demonstrated direct interaction between each DP and melanin. Except for dihydroxyphenylacetic acid, reduction kinetics were consistent with a mechanism involving noninteractive binding of both DP and ferricyanide to melanin prior to coupled electron transfer through the melanin backbone. Kinetic analysis afforded KB, a thermodynamic constant (M-1) for DP-melanin binding, and k', a rate parameter (M s-1) for electron transfer. A dimensionless enhancement factor (EF) was defined as k'KB/ks, with ks a pseudo-first-order constant (s-1) for ferricyanide reduction in the absence of melanin. Depending on the reductant, melanin either retards (EF less than 1) or accelerates (EF greater than 1) the rate of ferricyanide reduction. There appears to be a direct relationship between EF and depigmenting potency. There is no relationship between depigmenting power and the ability per se of the DP to bind to melanin or to reduce ferricyanide.

Potassium ferricyanide-elicited reactivation of steroid hydroxylase activities, in hepatic microsomes from SKF 525-A-induced male rats, was used as an indicator of complex formation between individual cytochrome P-450 isozymes and the SKF 525-A metabolite. Induction of male rats with SKF 525-A (50 mg/kg for three days) led to apparent increases in androst-4-ene-3,17-dione 16 beta- and 6 beta-hydroxylation to 6.7- and 3-fold of control activities. Steroid 7 alpha-hydroxylase activity was decreased to 0.8-fold of control and 16 alpha-hydroxylation was unchanged. Ferricyanide-elicited dissociation of the SKF 525-A metabolite-P-450 complex revealed an even greater induction of 16 beta- and 6 beta-hydroxylase activities (to 1.8- and 1.6-fold of activities in the absence of ferricyanide). Androst-4-ene-3,17-dione 16 alpha-hydroxylase activity increased 2-fold after ferricyanide but 7 alpha-hydroxylase activity was unaltered. An antibody directed against the male-specific cytochrome P-450 UT-A decreased androst-4-ene-3,17-dione 16 alpha-hydroxylase activity to 13% of control in hepatic microsomes from untreated rats. In contrast, 16 alpha-hydroxylase activity in microsomes from SKF 525-A-induced rats, before and after dissociation with ferricyanide, was reduced by anti UT-A IgG to 32 and 19% of the respective uninhibited controls. Considered together, these observations strongly suggest that the phenobarbital-inducible cytochrome P-450 isozymes PB-B and PCN-E are present in an inactive complexed state in microsomes from SKF 525-A-induced rat liver. Further, the increased susceptibility of androst-4-ene-3,17-dione 16 alpha-hydroxylase activity to inhibition by an antibody to cytochrome P-450 UT-A, following ferricyanide treatment of microsomes, suggests that this male sexually differentiated enzyme is also complexed after in vivo SKF 525-A dosage. In contrast, the constitutive isozyme cytochrome P-450 UT-F, which is active in steroid 7 alpha-hydroxylation, does not appear to be complexed to any extent in microsomes from SKF 525-A-induced rats.

Combinations of low concentrations of carbonyl cyanide p -trifluoromethoxyphenylhy-drazone (FCCP) with suboptimal concentrations of Dio-9, phloridzin, ajmaline, and dihydrodiscarine B synergistically inhibited cyclic and noncyclic photophosphorylation in spinach chloroplasts but their effects on the light-triggered ATPase were additive rather than synergistic. The effect was reversed by washing and prevented by dithioerythritol and by cistein. Carbonyl cyanide m -chlorophenylhydrazone (CCP) could replace FCCP but uncouplers of other types like atebrin did not substitute for FCCP. Combinations of FCCP with the four inhibitors synergistically uncoupled ferricyanide reduction in the presence of ADP and P i but not in their absence. The synergistic uncoupling was not observed on the light-dependent pH rise of chloroplast suspensions. Association of FCCP with any of the inhibitors completely abolished the stimulation of proton uptake or the inhibition of electron transport induced by low concentrations of ATP. This synergistic and peculiar uncoupling can not be ascribed to a modification of membrane permeability. One possible explanation is that the effect requires a conformational state of the membrane-bound coupling factor 1 (CF 1 ) induced by phosphorylating conditions which would facilitate the interaction of inhibitors and FCCP with the membrane.

Melanin synthesized from mushroom tyrosinase and 3,4-dihydroxyphenylalanine has been shown to oxidize NADH and NADPH, reduce ferricyanide, oxidized forms of cytochrome c and dichlorophenolindophenol, and catalyze the coupled oxidation of NADH and reduction of ferricyanide. Kinetic studies involving the determination of initial velocity at various concentrations of substrates and product inhibition measurements have been carried out on the NADH-ferricyanide-melanin reaction. The results are consistent with a ping-pong mechanism in which one product is formed prior to the reaction of melanin with the second substrate involving the reversible oxidation and reduction of melanin during the reaction. It may be concluded that melanin is capable of acting as an electron transfer agent in several reduction-oxidation systems.

Hen liver microsomes contained 0.20 nmol of cytochromeb5 per mg of protein. Upon addition of NADH about 95% cytochrome b5 was reduced very fast with a rate constant of 206 s−1 When ferricyanide was added to the reaction system the cytochrome stayed in the oxidized form until the ferricyanide reduction was almost completed. The reduced cytochrome b5 in microsomes was oxidized very rapidly by ferricyanide. The rate constant of 4.5 × 108 m −1 s−1, calculated on the basis of assumption that ferricyanide reacts directly with the cytochrome, was found to be more than 100 times higher than that of the reaction between ferricyanide and soluble cytochrome b5. To explain the results, therefore, the reverse electron flow from cytochrome b5 to the flavin coenzyme in microsomes was assumed. By three independent methods the specific activity of the microsomes was measured at about 20 nmol of NADH oxidized per s per mg of protein and it was concluded that the reduction of the flavin coenzyme of cytochrome b5 reductase by NADH is rate-limiting in the NADH-cytochrome b5 and NADH-ferricyanide reductase reactions of hen liver microsomes. In the NADH-ferricyanide reductase reaction the apparent Michaelis constant for NADH was 2.8 μ m and that for ferricyanide was too low to be measured. In the NADH-cytochrome c reductase reaction the maximum velocity was 2.86 nmol of cytochrome c reduced per s per mg of protein and the apparent Michaelis constant for cytochrome c was 3.8 μ m .

Delayed fluorescence (delayed light emission) from chloroplasts is increased by ATP, ADP and, to a lesser extent, by ITP. However, neither phosphorylation nor ATP utilization seems to play any part in the phenomenon since the energy transfer inhibitor deoxyphlorizin, which is also an ATPase inhibitor, has no effect on the enhancement of delayed fluorescence. The enhancement of delayed fluorescence by these nucleotides is accompanied by an increase in the extent of proton uptake and n decrease in the nonphosphorylating (basal) electron transport. Uncouplers and ionophores such as imidazole, glycineamide, morpholine, methyl-amine, cyclohexylamine, atebrin, and gramicidin nearly abolish delayed fluorescence. However, ammonium salts are exceptional; they considerably enhance the emission although they also abolish phosphorylation and proton gradient formation. This enhancement of delayed fluorescence occurs only near or above pH 8 and seems to be specific for ammonia when relatively intact lamellae are employed. When particles prepared therefrom with digitonin are used, methylamine also enhances the delayed fluorescence. The enhancement by ammonium salts is correlated with the uptake of ammonium ions. Valinomycin, which is known to increase the permeability of membranes to ammonium ions, abolishes delayed fluorescence in the presence of ammonium salts. It is suggested that (a) ammonia uncoupling abolishes the pH component of the light-induced transmembrane electrochemical potential gradient, but that (b) at higher pH's the electrical component of the gradient (the membrane potential) is not abolished and may even increase while (c) this increased membrane potential is responsible for enhancement of the delayed fluorescence. Gradients which contribute to delayed fluorescence are not necessarily capable of supporting phosphorylation. The requirements for phosphorylation seem more stringent than the requirements for delayed fluorescence and it may be that phosphorylation, unlike the delayed light emission, has an obligatory requirement for a pH gradient.

A spin-labeled hydrocarbon molecule having a lipid-water solubility such that both polar and apolar signals could be observed in chloroplast suspensions was used to monitor effects of light excitation. In glutaraldehyde-fixed chloroplasts, effects of light-induced swelling and shrinking were ascribed to a lipid structural change comparable to a temperature shift of 2°C. Under similar conditions freeze-fracture electron microscopy revealed changes in average sizes and clustering of membrane particles. Hence, the particle-free areas of the membrane interior, presumed to be the lipid domains, undergo changes in organization when chloroplasts are illuminated.

The conditions for steady-state Signal IIf formation in response to single turnover flashes in Tris-treated, 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU)-inhibited chloroplasts have been investigated. DCMU inhibits Signal IIf generation as the photoinactive state, Z P680 Q-A, accumulates. Potassium ferricyanide relieves this inhibition so that Signal IIf can be fully developed on each flash in a flash series. The effectiveness of ferricyanide in stimulating Signal IIf formation is dependent on its concentration, the flash repetition rate, and the salt composition of the chloroplast suspension. There are two models in the literature for Q-A oxidation under these inhibitory conditions: direct oxidation of Q-A by exogenous acceptors like ferricyanide or oxidation of Q-A by an endogenous acceptor, AH, which has a midpoint potential of approximately 400 mV. It is found that the direct exogenous acceptor model accounts well for these data, whereas the AH model does not explain several of these results. The apparent rate constant for the direct oxidation of Q-A by ferricyanide at various concentrations of salt has been calculated from our electron paramagnetic resonance (EPR) data and compared with the corresponding rate constant determined by S. Itoh from fluorescence data (Biochim, Biophys. Acta 504, 324-340, 1978); good agreement is found for the two different experimental approaches.

Two sites in the photosynthetic electron transport chain of spinach chloroplasts are sensitive to inhibition by the plastoquinone antagonist dibromothymoquinone (2,5-dibromo3-methyl-&isopropyl-p-benzoquinone). This compound imposes maximal inhibition on reactions involving electron transport from water to a terminal acceptor such as ferricyanide at concentrations of about 1 PM. At concentrations of about 10 PM, dibromothymoquinone also inhibits electron transport reactions catalyzed by photosystem II in the presence of pphenylenediimines or p-benzoquinones. This inhibition is observed in both untreated and KCN/Hg-inhibited chloroplast preparations. Thiol incubation of chloroplasts exposed to dibromothymoquinone relieves inhibition at both sites. This reversal of inhibition is, however, different for the two sites. Restoration of ferricyanide reduction, which is blocked by 1 pM dibromothymoquinone, required high thiol/inhibitor ratios and incubation times with thiol of up to 3 min. The reversal of inhibition of p-phenylenediimine reduction by photosystem II, on the other hand, requires a thiol/inhibitor ratio of 1, and incubation times as short as 5 s. Addition of bovine serum albumin to absorb dibromothymoquinone results in a partial restoration of photosystem II reactions, but ferricyanide reduction, which requires photosystem II and photosystem I, cannot be restored by this procedure.

Diethylstilbestrol reversibly inhibits electron transfer in particle-bound mitochondrial NADH-dehydrogenase (Keilin-Hartree preparation) at a site located in the vicinity of, or superimposed on, the piericidin A- and rotenone-specific sites. At this site, diethylstilbestrol half-maximal inhibitory concentration varies from 0.2 μ m (at 37 μ m NADH) to 2.6 μ m (at 180 μ m or higher NADH concentrations). The postulated localization of the diethylstilbestrol site is supported by (a) the similar effects of the diethylstilbestrol, piericidin A, and rotenone on the kinetics of the NADH-induced changes of the 470–500-nm chromophore; (b) the lack of action of diethylstilbestrol on the NADH-ferricyanide reductase and the NADH-acetylpyridine-adenine dinucleotide transhydrogenase reactions; (c) the lack of action of diethylstilbestrol on the NADH-2,3-dimethoxy-2′-methyl-1,4-benzoquinone reaction at infinite concentration of UQ 0 ; (d) the similar inhibitions of the NADH-UQ 2 reaction and NADH-cytochrome b k reaction; (e) the noncompetitive inhibition of the NADH-UQ 2 reaction; (f) the similar effects of diethylstilbestrol and rotenone on the kinetics of the NADH-UQ 0 reaction; and (g) the inhibition of the NADH-cytochrome b k reaction, in contrast with the insensitivity of the succinate-cytochrome b k reaction. The interaction of diethylstilbestrol with the NADH-dehydrogenase protein is supported by the summation of diethylstilbestrol, piericidin A, rotenone, and p -chloromercuribenzoate inhibitions. Diethylstilbestrol inhibition involves specific structural requirements as shown by the lesser (if any) inhibitory potency of hexestrol, diethylstilbestrol monomethyl ether, diethylstilbestrol dimethyl ether, and hexestrol dimethyl ether.

The enzymic activity of testis microsomes which mediates cleavage of the 2-carbon side chain from the 17-position of 21-carbon steroids is a mixed-function oxidase and recent reports have suggested that cytochrome P -450 is a participant in this reaction. The studies reported in this communication were intended to demonstrate that the flavoprotein, NADPH cytochrome c reductase, is also a participant in the reaction. The cleavage activity (referred to here as 17,20-lyase) from rat testis microsomes was shown to be inhibited by a number of agents which are electron acceptors for NADPH cytochrome c reductase. This finding indicates that the lyase activity is inhibited by diversion of electron flow and, more specifically, that the point of interruption is at the reductase. Cytochrome c , itself, is a noncompetitive inhibitor of lyase activity when NADPH is substrate. Lyase and reductase activity were diminished to an equal extent by heating a microsomal suspension. An antibody to cytochrome e reductase was prepared after purification of the reductase from rat liver. This antibody caused a parallel and equal reduction of activity of the lyase and reductase. These data show that the reductase and lyase activities are closely linked and suggest that the reductase functions as an electron carrier for the reduction of P -450.

The possible significance of carbon-centered radicals in hydrazine-induced carcinogenesis is explored by studies of the interaction between the 2-phenylethyl radical and DNA. The radical is efficiently generated during oxidation of phenelzine (2-phenylethyl-hydrazine) promoted by oxyhemoglobin or ferricyanide, as demonstrated by spin-trapping experiments and analysis of the reaction products. In the ferricyanide promoted oxidation, ethylbenzene formation accounts for about 40% of the initial drug concentration, from 5 to 100 m m phenelzine. By contrast, product formation in the presence of oxyhemoglobin depends on the enzyme concentration due to the fact that the prosthetic heme is destroyed during catalytic turnover. Covalent binding of the 2-phenylethyl radical to oxyhemoglobin is demonstrated by experiments with 2-[ 3 H]phenelzine, where tritium incorporation to the protein is inhibited by the spin-trap, α-(4-pyridyl-1-oxide)- N - tert -butylnitrone. The 2-phenylethyl radical is also able to alkylate DNA as suggested by electrophoretic studies with plasmid DNA, and proved by experiments with 2-[ 3 H]-phenelzine. The carbon-centered radical has a preference for attacking guanine residues as demonstrated by the use of sequencing techniques with 32 P-DNA probes. The results indicate that the 2-phenylethyl radical is an important product of phenelzine oxidation and that this species can directly damage protein and DNA.

The mechanistic stoichiometry for vectorial H + ejection coupled to electron transport through energy-conserving segments 1 + 2 was determined on cyanide-inhibited mitochondria from rat liver, rat heart, and Ehrlich ascites tumor cells, and on rat liver mitoplasts with ferricyanide or ferricytochrome c as electron acceptors. K + (+ valinomycin) and Ca 2+ were employed as permeant cations. Three different methods were employed. In the first, known pulses of ferricyanide were added, and the total H+ ejected was determined with a glass electrode. Such measurements gave H + 2 e − values exceeding 7.0 for both normal and tumor mitochondria with β-hydroxybutyrate and other NAD-linked substrates; uptake of Ca 2+ was also measured and gave the expected q + 2 e − ratios. The second type of measurement was initiated by addition of ferricytochrome c to rat liver mitoplasts, with H + ejection monitored with the glass electrode and ferricytochrome c reduction by dual-wavelength spectrophotometry; the H + 2 e − ratios generally exceeded 7.0. In the third type of measurement, mixing and dilution artifacts were eliminated by oxidizing ferrocytochrome c in situ with a small amount of ferricyanide. H + 2 e − ratios for rat liver mitoplasts oxidizing β-hydroxybutyrate consistently approached or exceeded 7.5. Over 150 measurements made under a variety of conditions gave observed H + 2 e − ejection ratios significantly exceeding 7.0, which correlated closely with H + 2 e − measurements on sites 1 + 2 + 3, sites 2 + 3, and site 2. Factors leading to the deficit of the observed ratios from the integral value 8 for sites 1 + 2 were discussed.

Two forms of cytochrome 559 have been demonstrated in chloroplasts, one of which is reducible by ascorbate (the A-form) and one of which cannot be reduced by ascorbate (the D-form). Analysis of the effects of temperature on the reduction of the cytochromes by light showed that the D-form was only observed between 20 and 40 °C. Below this range, only the A-form of the cytochrome was affected by light; the D-form remained oxidized. Also both electron transport and PMS+-photophosphorylation rates showed a sharp temperature transition at about 20 °C. Both electron transport and photophosphorylation are most sensitive to the inhibitor, CMU, at temperatures above 20 °C, and this coincides with the reduction of cytochrome 559 (D) which is extremely sensitive to CMU. Also, a transition temperature at 18 °C for fluorescent quenching was demonstrated.

N -methylglutamate dehydrogenase has been “solubilized,” by treatment with Triton X-100, and purified about fivefold. Eight different N -methyl amino acids serve as substrates for the enzyme with K m varying from approximately 0.2 m for the poorest substrate, sarcosine, to 54 μ m for the best substrate, N -methyl l -glutamate. The maximal velocity is independent of the N -methyl amino acid, yet decreases more than tenfold when the electron acceptor is changed from phenazine methosulfate to potassium ferricyanide. Double reciprocal plots measuring the effect of 2,6-dichloro-phenolindophenol on the K m of N -methyl l -glutamate show a series of parallel lines. The same result was obtained when sarcosine was used in place of N -methyl l -glutamate. These results are interpreted in terms of a two-step mechanism involving a reduced enzyme intermediate. The lack of product inhibition by glutamate or formaldehyde (or an equimolar mixture of the two) is discussed in terms of the possibility that N -methylene glutamate rather than glutamate or formaldehyde is the true product of the reaction.

Various anions when present at low concentrations in the acid stage of an acid-base phosphorylation experiment cause severe inhibition of subsequent ATP formation. Polyvalent anions, such as heparinate, polygalacturonate, polyvinylsulfate, and ferri- or ferrocyanide are most effective; sulfate is much less effective and chloride ions the least so. Brief pretreatment with polyanions at low pH also inhibits Mg-dependent ATPase of chloroplasts activated by acid-base transition, and light-dependent proton uptake and photophosphorylation supported either by cyclic or noncyclic electron flow. A similar sensitivity of the Ca-dependent ATPase activity of solubilized chloroplast coupling factor, and preliminary evidence for its aggregation under these conditions, suggests denaturation of this enzyme as one basis for the action of polyanions. Further evidence indicates that the coupling factor, before or after denaturation, is detached from the chloroplast thylakoid membranes.

Methylamine-induced cells of Pseudomonas MA (ATCC No. 23819) contain a particulate enzyme system which catalyzes the reaction: 2 N -methyl l -glutamate + O 2 → 2 l -glutamate + 2 formaldehyde + (H 2 O). The stoichiometry of this reaction (2 moles N -methyl l -glutamate utilized per mole of oxygen) suggests that the enzyme is a dehydrogenase. During the oxidation of N -methyl l -glutamate, cytochromes b and c are enzymatically reduced, supporting the conclusion that the enzyme is a respiratory chain-linked dehydrogenase. It is proposed that the function of this enzyme is to activate the methyl group of N -methyl l -glutamate so that it may be further metabolized by this microorganism.

1. 1. The quantum requirement for photolysis with either whole or lyophilized Chlorella cells is 19.0 with either ferricyanide or quinone as oxidant. 2. 2. The quantum requirement of photolysis with Chlorella cells is independent of the wavelength of the irradiating source. 3. 3. The use of lyophilized Chlorella cells has been introduced for investigating photolysis.

Divalent cations have previously been shown to affect chloroplast electron transport and phosphorylation. Here, a new effect of divalent cations on high-energy states of chloroplasts is reported. It was found that when washed chloroplasts were suspended in sucrose, high (uncoupled) rates of electron flow were observed unless low concentrations of salts of monovalent (approximately 1 m m ) or divalent (approximately 20 μ m ) cations were added. The inhibition of the high rate of electron flow induced by divalent cations was reversed by uncouplers. Higher rates of ATP formation were observed when electron flow was controlled by cations than when it was not so controlled. Cations increased the intensity of delayed light and the extent of the 515-nm absorption change; two parameters which reflect in some way the efficiency of energy conservation. In contrast, cations had little effect on the rate of uptake or decay of H + ions or on the total extent of the proton gradient, suggesting that they act on an earlier intermediate. These results indicate that the presence of cations profoundly affects the coupling of the energy available in redox reactions to the energy-conserving mechanism in the chloroplast membrane.

The cytochromes b and b T of pigeon heart mitochondria have half-reduction potentials ( Em 's) of +30 mV and −30 mV at pH 7.2. The midpoint potentials of these cytochromes become more negative by 30–60 mV per pH unit when the pH is made more alkaline. Detergents may be used to prepare a succinate-cytochrome c reductase free of cytochrome oxidase in which the activation of electron transport induced by oxidation of cytochrome c 1 causes the half-reduction potential of cytochrome b T to become at least 175 mV more positive than in the absence of electron transport. This change is interpreted as indicating that the primary energy conservation reaction at site 2 remains fully functional in the purified reductase. Preliminary electron paramagnetic resonance spectra of the succinate-cytochrome c reductase as measured at near liquid helium temperatures are presented.

H 2 O 2 production by coupled mitochondrial fractions from the protozoan, Crithidia fasciculata , has been measured spectrophotometrically by the formation of the stable enzyme-substrate complex with yeast cytochrome c peroxidase. H 2 O 2 formation was observed with succinate, l -α-glycerophosphate, l -proline, α-ketoglutarate, and with endogenous substrate. The maximum rate of H 2 O 2 generation obtained with each substrate in the presence of antimycin A was about 10% of the state 4 rate of O 2 respiration, and only 1–2% of the carbonylcyanide m -fluorophenylhydrazone-uncoupled respiratory rate. Therefore, excess O 2 uptake due to the formation of H 2 O 2 cannot satisfactorily account for the low ADP:O ratios previously reported. Cytochrome c peroxidase activity was measured in mitochondrial preparations by recording the decrease in absorbance at 550 nm during the oxidation of horse heart ferrocytochrome c which was observed after addition of H 2 O 2 . The distribution of activity after sonic disruption of mitochondrial preparations was that expected for a soluble enzyme. The activity was proportional to the amount of enzyme protein added, and was abolished by heating at 100 °C for 3 min. Total cytochrome c peroxidase activity in mitochondrial fractions isolated from C. fasciculata was calculated to be 0.3% that of isolated yeast mitochondria, but it is suggested that the in vivo activity may be considerably higher than this estimate.

DPNH dehydrogenase, solubilized from a particulate DPNH oxidase by treatment of the latter with ethanol at pH 4.8 and 43 °, has been further purified by precipitation with ammonium sulfate at pH 7.8 and by filtration through Sephadex G-25. The enzyme migrates as a single band when subjected to electrophoresis on starch gel or polyacrylamide. Based upon a molecular weight of 70,000 (determined by filtration through Sephadex G-200), each molecule of protein contains 1 FMN, 2 nonheme irons, 1 labile sulfide group, and 7 thiol groups. The latter are determined by titration of the protein with DTNB in the presence of 4 M urea. When the enzyme is reduced by DPNH under anaerobic conditions and in the absence of an external electron acceptor, the flavin appears to be fully reduced rather than at the semiquinone level; under these conditions, each mole of enzyme oxidizes approximately 3 moles of DPNH. Removal of the bound FMN by passage of the enzyme through Florisil or Bio-Gel does not affect the DPNH-ferricyanide activity while completely abolishing activity with cytochrome c or 2,6-dichlorophenolindophenol; cytochrome c activity can be largely restored by the addition of FMN. p-Chloromercuribenzoate (pCMB) inhibits the dehydrogenation of DPNH coupled to ferricyanide, indophenol, or cytochrome c; inhibition of the DPNH-ferricyanide activity is a biphasic function of pCMB concentration. Preincubation of the enzyme with DPNH increases the sensitivity toward pCMB when ferricyanide and cytochrome c are used as acceptors.

Nitrobacter agilis contains at least five cytochromes. These include cytochrome c ( E m7.0 + 274 mV), cytochrome a ( E m7.0 + 240 mV), cytochrome a 3 ( E m7.0 + 400 mV) and two cytochromes ( E m7.0 + 352 mV and E m7.0 + 100 mV) with an absorbance band at 590 nm which is usually attributed to cytochrome a 1 . Although all of these cytochromes are rapidly oxidized when oxygen is added to an anaerobic suspension of cell-free extracts, the terminal oxidase is cytochrome a 3 . The properties of the cytochromes designated a and a 3 are similar to those of mitochondrial cytochromes a and a 3 .

Dialysis affects the shape of hemoglobin redox titration curves, thus leading to different end point determinations. At pH 7.9, the oxidation of ferrohemoglobin by ferricyanide is not a simple reversible reaction but appears to proceed through an irreversible stepwise mechanism.

Broken cell preparations of Rhodospirillum rubrum have been obtained by subjecting the cells to the combined action of lysozyme and osmotic shock. These lysed preparations incorporate radioactivity from p-hydroxybenzoate-U-14C and acetate-2-14 C into 2-polyprenylphenols and ubiquinones. They also incorporate methionine(methyl-14C) and S-adenosylmethionine-(methyl-14C) into ubiquinones and 2-decaprenyl-6-methoxyphenol. Relatively cell-free extracts were also obtained by sonicating Rhodospirillum rubrum cells. These extracts were efficient in incorporating p-hydroxybenzoate-U-14C into 2-polyprenylphenols. Although ubiquinone synthesis was occasionally observed in both preparations it could be accounted for by the presence of small numbers of unbroken cells in the extract which were detected by means of viable cell counts. These cells were not sufficient to account for the vast majority of 2-polyprenylphenol synthesis and were virtually absent from sonicates capable of synthesizing substantial amounts of 2-polyprenylphenol after centrifugation for 1 hr at 48,000g. The incorporation of p-hydroxybenzoate-U-14C into 2-polyprenylphenol by both types of extract is greatly stimulated by the addition of a source of side-chain precursor in the form of a crude extract of Micrococcus lysodeikticus cells and isopentenyl pyrophosphate. Cysteine and ascorbic acid stimulate this conversion of p-hydroxybenzoate to 2-polyprenylphenol. Attempts to stimulate ubiquinone synthesis in the broken cell preparations by the use of various redox reagents (dichlorobenzenoneindophenol, phenazine methosulfate, methylene blue, neotetrazolium chloride, triphenyltetrazolium bromide, cytochrome c) either in catalytic quantities or as terminal electron acceptors, failed to restore ubiquinone synthesis; most of the reagents proved to be extremely inhibitory to 2-polyprenylphenol synthesis. The R. rubrum extracts, especially when supplemented with Micrococcus extract and isopentenyl pyrophosphate, incorporate radioactivity from p-hydroxybenzoate-U-14 C into another intermediate in ubiquinone synthesis. p-Hydroxybenzoate-7-14 C is also incorporated into this compound, but not methionine-(methyl-14C). The sonicated preparations of R. rubrum are capable of converting this compound to 2-polyprenylphenol. On this basis a reasonable structure to assign to this compound is 3-polyprenyl-4-hydroxybenzoate. A bacterium, found as a contaminant in some cultures of R. rubrum, has been isolated and shown to incorporate phydroxybenzoate-U-14 C into ubiquinone. The organism, when incubated anaerobically with phydroxybenzoate-U-14 C (or p-hydroxybenzoate-7-14C) in the presence of potassium ferricyanide, accumulated 3-polyprenyl-4-hydroxybenzoate. When the cells were washed and the p-hydroxybenzoate-U-14C was diluted with unlabeled p-hydroxybenzoate, subsequent aerobic incubation of the cells converted almost all the 3-polyprenyl-4-hydroxybenzoate to ubiquinone. The distribution of prenylogues of 2-polyprenylphenol in R. rubrum extracts supplemented with MLE and IPP is the same as that observed in whole cells.

Trehalose-6,6′-dimycolate (cord factor), a toxic glycolipid of Mycobacterium tuberculosis , inhibited the phosphorylation coupled to the oxidation of either succinate or NADH-linked substrates and caused a loss of respiratory control in mouse liver mitochondria. Preincubation of the mitochondrial suspension with cord factor was necessary for this inhibitory action. Liver mitochondria from a variety of animal species were susceptible to the action of cord factor. Phosphorylation coupled to the oxidation of succinate by ferricyanide (site II phosphorylation) was completely damaged, while that associated with the oxidation of ascorbate-TMPD (site III phosphorylation) was not affected by cord factor. Mycolic acid, either alone or with trehalose, acetylated cord factor, and a nontoxic synthetic cord factor analog, 6, x -di-(3′-acetoxy, x -methoxymycolanoyl)- N -acetyl- d -glucosamine, were without effect, while two toxic cord factor analogs, methyl 6-(3′, x -sulphitomycolanoyl)-α- d -glucoside and 6-(3′-acetoxy, x -methoxymycolanoyl)- N -acetyl- d -glucosamine, affected the mitochondrial oxidative phosphorylation as did cord factor. Cord factor stimulated slightly the mitochondrial ATPase. The effect of cord factor and DNP to induce the mitochondrial ATPase was additive. The 14 C-labeled cord factor was not firmly bound to mitochondria.

Human red blood cell ghosts (RBCG) prepared by gradual osmotic lysis (Danon procedure) down to 30 mOsm (G-30) were exposed to a 9 mOsm hypotonic treatment (G-9) which increased NADH-ferricyanide reductase activity by 272% and the (Mg + Na + K) ATPase activity by 40%. Sonic oscillation of G-30 did not affect ATPase but increased NADH oxidation 7 times. After fragmentation the entire NADH ferricyanide activity remained bound to membrane particles and showed the same specificity for electron acceptors and donors. The effect of temperature and divalent ions on the NADH oxidation was also studied in intact arid fragmented ghosts. The results indicate that permeability to the substrates and the vectorial assembly of active sites of the enzyme are the rate limiting factors of this reaction. Histochemical observation of the erythrocyte ghosts, using tetrazolium salt (NBT) as electron acceptor, allowed us to locate the enzyme on the membrane. The effect of detergents, phospholipases A and C, and organic solvents on the NADH: (acceptor)oxidoreductase and ATPase showed: (a) complete inactivation of both enzymes at 0.5% sodium dodecyl sulphate; (b) phospholipases A and C inhibited (Mg + Na + K)ATPase but not NADH oxidation; (c) treatment of freeze-dried membrane particles with anhydrous organic solvents such as acetone, pentane or butanol did not affect either of two enzymic activities, but a gradual inactivation occurred upon aqueous-acetone treatment. The NADH: (acceptor)oxidoreductase was dissociated from the phospholipidprotein complex by an aqueous butanol extraction after the removal of loosely bound lipid with anhydrous butanol. These results indicate that NADH: (acceptor)oxidoreductase of RBCG as well as (Mg + Na + K)-ATPase is a structural enzyme of these membranes.

Isolated pea chloroplasts were washed once in 10 m m NaCl and were then suspended in “low-salt” medium. Approximately one-half of the photosystem II reaction centers of these salt-depleted membranes were found to be photochemically inactive. These units became active in the presence of low concentrations of divalent cations (5–10 m m Mg 2+ ) or high concentrations of monovalent cations (150–200 m m Na + ), as evidenced by a twofold increase in the steady-state flash yield of oxygen evolution under short (~10-μs) saturating repetitive flashes (two per second). The half-maximal increase in flash yield occurred at ~2 mM Mg 2+ or ~75 m m Na + . The flash yield of hydroxylamine oxidation in these low-salt chloroplasts increased twofold after Mg 2+ addition, indicating that the cation action was close to the reaction-center chlorophyll complex. The relation between flash yield and dark time between flashes was not changed significantly by Mg 2+ , indicating that the rate-limiting step of the overall electron transport (H 2 0 —→ ferricyanide) was not affected significantly. When the rate-limiting step was bypassed using silicomolybdate as the photosystem II electron acceptor (in the presence of diuron), the reduction rate doubled in the presence of Mg 2+ , even under continuous, saturating light. In glutaraldehyde-fixed chloroplasts, Mg 2+ did not increase the flash yield of O 2 evolution; this suggests that protein conformational changes in the chloroplast membranes were involved in Mg 2+ activation of photosystem II centers.

Simultaneous oxidation-reduction potential versus absorbance titrations on intact mitochondria have allowed us to measure the individual spectral and thermodynamic properties of cytochromes a and a 3 in the presence or absence of added ligands. In coupled pigeon heart mitochondria, saturated with CO, the reduced a 3 CO compound can be fully oxidized by ferricyanide under conditions of high phosphate potential, i.e., on addition of ATP. These ATP-induced effects are reversed by the presence of oligomycin or uncouplers with complete restoration of the reduced a 3 CO compound. This observation has permitted us to examine the spectral and thermodynamic properties of the a 3 CO compound in some detail. The data indicate that, in the presence of ATP, the midpoint potential of the a 3 CO compound varies as a function of CO concentration in a thermodynamically predictable fashion. The ATP-induced changes on the a 3 CO compound are directly analogous to the changes reported earlier for cytochrome a 3 in the absence of added ligands.

Coenzymes Q10 and Q3 are incorporated into dipalmitoylphosphatidylcholine and egg yolk lecithin liposomes. Dithionite reduction of ferricyanide trapped inside these phospholipid vesicles is taken as a measure of ubiquinone-mediated transport of reducing equivalents. The reaction shows complex pattern with a high order for CoQ. The initial transport rates are very sensitive to the membrane physical state, being considerably reduced at temperatures below the phase transition of the pure dipalmitoylphosphatidylcholine, both for CoQ10 and CoQ3 reconstituted with this phospholipid. It is suggested that a different reaction mechanism operates in fluid and rigid membranes. This suggestion is related to the possible organization of CoQs in phospholipid membranes.