Characteristics of the interaction of adrenal lipoamide dehydrogenase with physiological and quinone electron acceptors

Lipoamide dehydrogenase (EC 1.6.4.3) from the ketoglutarate dehydrogenase complex of adrenals catalyzes the oxidation of NADH by lipoamide and quinone compounds according to the "ping-pong" scheme. The catalytic constants of these reactions are equal to 220 and 24 s-1, respectively (pH 7.0). The maximal quinone reductase activity is observed at pH 5.6, whereas the lipoamide reductase activity changes insignificantly at pH 7.5-5.5. The maximal dihydrolipoamide-NAD+ reductase activity is observed at pH 7.8. The oxidative constants of quinone electron acceptors vary from 6 X 10(6) to 4 X 10(2) M-1 s-1 and increase with their redox potential. The patterns of NAD+ inhibition in the quinone reductase reaction differ from that of lipoamide reductase reaction. The quinones are reduced by lipoamide dehydrogenase in the one-electron mechanism.     

Diaphorase reactions of lipoamide dehydrogenases from the adrenal ketoglutarate dehydrogenase complex

Lipoamide dehydrogenase, a component of the bovine adrenal ketoglutarate dehydrogenase complex, catalyzes the oxidation of NADH by p-quinones and ferricyanide. The kinetics of oxidation obey the ping-pong mechanism. At pH 7.0, the constants for the active center oxidation by quinones (kox) are equal to 1.1 X 10(4)-5.3 X 10(5) M-1s-1 and increase as the acceptor potential rises. The values of kox for quinones change insignificantly within the pH range of 7.7-5.0, whereas that for ferricyanide increases 10-fold with a decrease of pH from 7.0 to 5.0. The value of the catalytic constant for the enzyme (kcat) reaches its maximum at pH 5.5. The quinones interact with the thiol groups of lipoamide dehydrogenase by inhibiting the fluorescence of FAD and diaphorease activity. The reaction is catalyzed by a basic amino acid (pK 6.7) within the composition of the enzyme.     

Reduction of laccase type 1 copper by 3,4-dihydroxyphenylalanine and other catechol derivatives

3,4-Dihydroxyphenylalanine (DOPA) is not a preferred substrate of Rhus vernicifera laccase, as rate constants for the anaerobic reduction of the type 1 cupric atom by L-DOPA (6.3 X 10(1) M-1 s-1), D-DOPA (2.6 X 10(1) M-1 s-1), and L-DOPA methyl ester (2.6 X 10(1) M-1 s-1) are considerably smaller than k1 (catechol) (7 X 10(2) M-1 s-1) and rate constants characteristic of numerous other nonphysiological organic substrates (25 degrees C, pH 7.0, I = 0.5 M). The reactions of DOPA derivatives with laccase are unique, however, in that a two-term rate law pertains: kobsd = k0 + k1[phenol]; k0(L-DOPA) = 7 X 10(-2) s-1. The reactivities of other catechol derivatives (pyrogallol, gallic acid, and methyl gallate) with laccase type 1 copper were also examined.     

Reactivity of cuprous stellacyanin as a quinone and semiquinone reductase

The reactivity of cuprous stellacyanin as a quinone and semiquinone reductase has been examined. Rate constants (25.0 degrees C) measured for the oxidation of stellacyanin by 1,4-benzoquinone and benzosemiquinone are 2.3 X 10(4) M-1 s-1 (delta H not equal to = 4.4 kcal/mol, delta S not equal to = -24 eu) and 5.1 X 10(6) M-1 s-1, respectively [pH 7.0, I = 0.1 M (phosphate)]. The agreement of these rate constants with those calculated on the basis of relative Marcus theory is discussed. Stellacyanin is more effective than laccase in quenching benzosemiquinone, suggesting that the physiological role of this metalloprotein is to regulate the concentration of free radicals generated through the laccase-catalyzed oxidation of phenols.     

The DNA guanyl radical: kinetics and mechanisms of generation and repair

The one-electron oxidation of DNA bases and single-stranded DNA was studied by pulse radiolysis of aqueous solutions from pH 7-7.4 at 20 degrees C. Thallic ions, Tl(II), were found to rapidly oxidize the purine nucleotides, deoxyguanosine 5'-monophosphate, k[Tl(II) + dGMP2-] = 3.4.10(9) M-1.s-1, and deoxyadenosine 5'-monophosphate, k[Tl(II) + dAMP2-] = 1.3.10(8) M-1.s-1. The reactivities of Tl(II) ions with model pyrimidine DNA bases, 1-methylcytosine and 1-methylthymine, were too low to be measured by pulse radiolysis, k less than 10(7) M-1.s-1. The Tl(II)-mediated oxidation of ssDNA, k = 2.8.10(8) M-1.s-1, produces DNA-guanyl radical, DNA-G.(-H), exclusively. The DNA-guanyl radical is found to be a potent oxidant in neutral media, E7 = 1.04 +/- 0.05 V. It rapidly oxidizes the aromatic amino acids in glycyl-tryptophan and tyrosine methyl ester, k = 3.6.10(7) M-1.s-1 and k = 1.7.10(8) M-1.s-1, respectively. These electron transfer processes indicate that a positive 'hole' may be transferred from DNA to a DNA-associated protein. The positive 'hole' in DNA can also be repaired by antioxidants, which are electron donors. The chemical repair of the DNA-guanyl radical by negatively charged antioxidants is slower than that by positively charged and neutral antioxidants.     

The mechanism of the quinone reductase reaction of pig heart lipoamide dehydrogenase.

The relationship between the NADH:lipoamide reductase and NADH:quinone reductase reactions of pig heart lipoamide dehydrogenase (EC 1.6.4.3) was investigated. At pH 7.0 the catalytic constant of the quinone reductase reaction (kcat.) is 70 s-1 and the rate constant of the active-centre reduction by NADH (kcat./Km) is 9.2 x 10(5) M-1.s-1. These constants are almost an order lower than those for the lipoamide reductase reaction. The maximal quinone reductase activity is observed at pH 6.0-5.5. The use of [4(S)-2H]NADH as substrate decreases kcat./Km for the lipoamide reductase reaction and both kcat. and kcat./Km for the quinone reductase reaction. The kcat./Km values for quinones in this case are decreased 1.85-3.0-fold. NAD+ is a more effective inhibitor in the quinone reductase reaction than in the lipoamide reductase reaction. The pattern of inhibition reflects the shift of the reaction equilibrium. Various forms of the four-electron-reduced enzyme are believed to reduce quinones. Simple and 'hybrid ping-pong' mechanisms of this reaction are discussed. The logarithms of kcat./Km for quinones are hyperbolically dependent on their single-electron reduction potentials (E1(7]. A three-step mechanism for a mixed one-electron and two-electron reduction of quinones by lipoamide dehydrogenase is proposed.     

Reaction of complex I of the mitochondrial electron transport chain with artificial oxidizers

The steady-state kinetics of oxidation of the mitochondrial NADH: ubiquinone oxidoreductase (complex I, EC 1.6.99.3) by artificial electron acceptors--p-quinones and inorganic complexes has been investigated. A limiting stage in the NADH: ferricyanide reductase reaction is a reductive half-reaction. Ferricyanide interacts with negative-charged protein groups taking part in the NADH binding. The rate constants of the quinone reduction by complex I vary from 1.10(6) to 4.10(3) M-1s-1. The NADH, NAD+ and ADP-ribose inhibition data indicate that oxidizers in the rotenono-insensitive reaction interact with the redox centre near the NAD+/NADH binding site, most probably with FMN.     

On the origin of the lactate dehydrogenase induced rate effect

To evaluate the ability of lactate dehydrogenase to facilitate the bond making/breaking steps for both the addition of pyruvate enol to NAD (pyruvate adduct reaction) and the normal redox reaction, the ability of the enzyme to facilitate the tautomerization of bound pyruvate is assessed. In addition, the equilibrium constants for the adduct reaction are obtained for both bound and free reactants from the ratio of the rate constants in the forward and reverse reactions (at pH 7). The latter comparison indicates that the enzyme facilitates bond making/breaking in the (forward) pyruvate adduct reaction by a factor of about 10(11) M. Similar comparisons suggest that reactant immobilization accounts for about 1000 M of this 10(11) M rate effect. Since the (pH-independent) rate constant for the ketonization of bound pyruvate enol assisted by the external buffer, imidazolium ion, is 2 X 10(7) M-1 s-1 and the corresponding rate constant for free pyruvate enol, again assisted by imidazolium ion, is 35 M-1 s-1 [Burger, J. W., II, & Ray, W. J., Jr. (1978) Biochemistry 17, 1664], the enzyme facilitates the bond making/breaking steps associated with the conversion of bound HO-C less than to bound O = C less than by a factor of about 10(6)-fold. The product of the above two rate enhancement factors and the rate factor suggested previously for the environmental effect on NAD produced by its binding to lactate dehydrogenase, 100-fold, is 10(11) M, and it accounts for the bond making/breaking effects exerted by the enzyme in the pyruvate adduct reaction. The rate constant for oxidation of ethanol (a model for lactate) by 1-methylnicotinamide (a model for NAD) is about 5 X 10(-12) M-1 s-1 at 25 degrees C in pure ethanol (delta H for this reaction is about 30 kcal/mol). The ratio of the rate constants for E X NAD X Lac----E X NADH X Pyr and the above model reaction is estimated as about 10(14) M in water; i.e., the LDH-induced rate effect is about 10(14) M. The product of the values for the above rate factors for the normal redox reaction is about 10(12) M. Although the value of this product is less certain than that for the adduct reaction, these rate factors do account for much of the LDH-induced rate effect.     

Kinetic studies of calcium binding to parvalbumins from bullfrog skeletal muscle

In addition to steady-state properties of calcium binding to parvalbumins, kinetic studies are required for adequate evaluation of the physiological roles of parvalbumins. By using a dual-wavelength spectrophotometer equipped with a stopped-flow accessory, the transient kinetics of calcium binding to parvalbumins (PA-1 and 2) from bullfrog skeletal muscle was examined at 20 degrees C in medium containing 20 mM MOPS-KOH, pH 6.80, 0.13 mM tetramethylmurexide, 25 microM CaCl2, metal-deprived PA-1 or PA-2, various concentrations of Mg2+, and KCl to adjust the ionic strength of the medium to 0.106. The results can be explained in terms of the following rate constants under the conditions mentioned above when a second-order kinetic scheme is assumed. For PA-1, the association and apparent dissociation rate constants for Ca2+ are 1.5 X 10(7) M-1 X s-1 and 1.5 s-1, respectively, or more. The rate constants for Mg2+ are 7,500 M-1 X s-1 and 5-6 s-1, respectively. For PA-2, the rate constants for Ca2+ are 7 X 10(6) M-1 X s-1 and 1.16 s-1, respectively, and those for Mg2+ are 3,500 M-1 X s-1 and 3.5-4 s-1, respectively. Increased affinities for Ca2+ and Mg2+ at 10 degrees C are largely due to decreased apparent dissociation rate constants for these divalent cations, because no significant change in the association rate constants was found.     

Transient kinetic studies of substrate inhibition in the horse liver alcohol dehydrogenase reaction

The rate-limiting step of ethanol oxidation by alcohol dehydrogenase (E) at substrate inhibitory conditions (greater than 500 mM ethanol) is shown to be the dissociation rate of NADH from the abortive E-ethanol-NADH complex. The dissociation rate constant of NADH decreased hyperbolically from 5.2 to 1.4 s-1 in the presence of ethanol causing a decrease in the Kd of NADH binding from 0.3 microM for the binary complex to 0.1 microM for the abortive complex. Correspondingly, ethanol binding to E-NADH (Kd = 37 mM) was tighter than to enzyme (Kd = 109 mM). The binding rate of NAD+ (7 X 10(5) M-1s-1) to enzyme was not affected by the presence of ethanol, further substantiating that substrate inhibition is totally due to a decrease in the dissociation rate constant of NADH from the abortive complex. Substrate inhibition was also observed with the coenzyme analog, APAD+, but a single transient was not found to be rate limiting. Nevertheless, the presence of substrate inhibition with APAD+ is ascribed to a decrease in the dissociation rate of APADH from 120 to 22 s-1 for the abortive complex. Studies to discern the additional limiting transient(s) in turnover with APAD+ and NAD+ were unsuccessful but showed that any isomerization of the enzyme-reduced coenzyme-aldehyde complex is not rate limiting. Chloride increases the rate of ethanol oxidation by hyperbolically increasing the dissociation rate constant of NADH from enzyme and the abortive complex to 12 and 2.8 s-1, respectively. The chloride effect is attributed to the binding of chloride to these complexes, destabilizing the binding of NADH while not affecting the binding of ethanol.     

Reaction mechanism of the magnesium ion-dependent adenosine triphosphatase of frog muscle myosin and subfragment 1.

The Mg2+-dependent ATPase (adenosine 5'-triphosphatase) mechanism of myosin and subfragment 1 prepared from frog leg muscle was investigated by transient kinetic technique. The results show that in general terms the mechanism is similar to that of the rabbit skeletal-muscle myosin ATPase. During subfragment-1 ATPase activity at 0-5 degrees C pH 7.0 and I0.15, the predominant component of the steady-state intermediate is a subfragment-1-products complex (E.ADP.Pi). Binary subfragment-1-ATP (E.ATP) and subfragment-1-ADP (E.ADP) complexes are the other main components of the steady-state intermediate, the relative concentrations of the three components E.ATP, E.ADP.Pi and E.ADP being 5.5:92.5:2.0 respectively. The frog myosin ATPase mechanism is distinguished from that of the rabbit at 0-5 degrees C by the low steady-state concentrations of E.ATP and E.ADP relative to that of E.ADP.Pi and can be described by: E + ATP k' + 1 in equilibrium k' - 1 E.ATP k' + 2 in equilibrium k' - 2 E.ADP.Pi k' + 3 in equilibrium k' - 3 E.ADP + Pi k' + 4 in equilibrium k' - 4 E + ADP. In the above conditions successive forward rate constants have values: k' + 1, 1.1 X 10(5)M-1.S-1; k' + 2 greater than 5s-1; k' + 3, 0.011 s-1; k' + 4, 0.5 s-1; k'-1 is probably less than 0.006s-1. The observed second-order rate constants of the association of actin to subfragment 1 and of ATP-induced dissociation of the actin-subfragment-1 complex are 5.5 X 10(4) M-1.S-1 and 7.4 X 10(5) M-1.S-1 respectively at 2-5 degrees C and pH 7.0. The physiological implications of these results are discussed.     

Kinetics of the oxidation of reduced nicotinamide adenine dinucleotide by horseradish peroxidase compounds I and II

The transient state kinetics of the oxidation of reduced nicotinamide adenine dinucleotide (NADH) by horseradish peroxidase compound I and II (HRP-I and HRP-II) was investigated as a function of pH at 25.0 degrees C in aqueous solutions of ionic strength 0.11 using both a stopped-flow apparatus and a conventional spectrophotometer. In agreement with studies using many other substrates, the pH dependence of the HRP-I-NADH reaction can be explained in terms of a single ionization of pKa = 4.7 +/- 0.5 at the active site of HRP-I. Contrary to studies with other substrates, the pH dependence of the HRP-II-NADH reaction can be interpreted in terms of a single ionization with pKa of 4.2 +/- 1.4 at the active site of HRP-II. An apparent reversibility of the HRP-II-NADH reaction was observed. Over the pH range of 4-10 the rate constant for the reaction of HRP-I with NADH varied from 2.6 X 10(5) to 5.6 X 10(2) M-1 s-1 and of HRP-II with NADH varied from 4.4 X 10(4) to 4.1 M-1 s-1. These rate constants must be taken into consideration to explain quantitatively the oxidase reaction of horseradish peroxidase with NADH.     

Tight binding of divalent cations to monomeric actin. Binding kinetics support a simplified model

Using the fluorescent Ca2+ selective chelator Quin2 to induce and measure the dissociation of Ca2+ from actin, we have recently found that actin binds Ca2+ and Mg2+ much more tightly than previously thought (Gershman, L.C., Selden, L.A., and Estes, J.E. (1986) Biochem. Biophys. Res. Commun. 135, 607-614). In this report, we show that the kinetics of dissociation of Ca2+ from Ca-actin and Mg2+ from Mg-actin closely parallel the fluorescence changes in 1,5-I-N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine (AEDANS)-actin, suggesting that the 1,5-I-AEDANS-actin fluorescence directly reflects slow first-order cation exchange rather than a slow Mg2+-induced isomerization as originally proposed by Frieden (Frieden, C. (1982) J. Biol. Chem. 257, 2882-2886). Measuring divalent cation exchange directly, we have determined the dissociation rate constants for Ca2+ (k-Ca) and Mg2+ (k-Mg), the equilibrium dissociation constants for Ca2+ (KCa), and the ratio of cation binding affinities, KMg/Kca, to actin over the pH range 7-8. We have found that k-Ca is 5-10 times greater than k-Mg and KMg is about 4 times greater than KCa. From the data we calculate the association rate constants for Ca2+ (kCa) and Mg2+ (kMg) to be about 7 X 10(6) M-1 s-1 and 2 X 10(5) M-1 s-1, respectively. kCa appears to be diffusion-limited, but kMg is significantly smaller due to the characteristics of the Mg2+ aquo ion. These findings are consistent with a simple first-order binding model for the tight binding of divalent cations to actin.     

Two-electron reduction of quinones by Enterobacter cloacae NAD(P)H:nitroreductase: quantitative structure-activity relationships

Enterobacter cloacae NAD(P)H:nitroreductase (NR; EC 1.6.99.7) catalyzes two-electron reduction of a series of quinoidal compounds according to a "ping-pong" scheme, with marked substrate inhibition by quinones. The steady-state catalytic constants (k(cat)) range from 0.1 to 1600s(-1), and bimolecular rate constants (k(cat)/K(m)) range from 10(3) to 10(8)M(-1)s(-1). Quinones, nitroaromatic compounds and competitive to NADH inhibitor dicumarol, quench the flavin mononucleotide (FMN) fluorescence of nitroreductase. The reactivity of NR with single-electron acceptors is consistent with an "outer-sphere" electron transfer model, taking into account high potential of FMN semiquinone/FMNH(-) couple and good solvent accessibility of FMN. However, the single-electron acceptor 1,1(')-dibenzyl-4,4(')-bipyridinium was far less reactive than quinones possessing similar single-electron reduction potentials (E(1)(7)). For all quinoidal compounds except 2-hydroxy-1,4-naphthoquinones, there existed parabolic correlations between the log of rate constants of quinone reduction and their E(1)(7) or hydride-transfer potential (E(7)(Q/QH(-))). Based on pH dependence of rate constants, a single-step hydride transfer seems to be a more feasible quinone reduction mechanism. The reactivities of 2-hydroxy-1,4-naphthoquinones were much higher than expected from their reduction potential. Most probably, their enhanced reactivity was determined by their binding at or close to the binding site of NADH and dicumarol, whereas other quinones used the alternative, currently unidentified binding site.     

The oxidation of ferrocytochrome c in nonbinding buffer.

The apparent equilibrium constant and rate of oxidation was investigated for the reaction of cytochrome c with iron hexacyanide. It was found that if horse heart ferricytochrome c was exposed to ferricyanide (to oxidize traces of reduced protein) the cytochrome subsequently, even after extensive dialysis, had an apparent equilibrium constant different from that of electrodialyzed protein. The effect of ferricyanide ion apparently cannot be removed by ordinary dialysis. The ionic strength dependence of the apparent equilibrium constant and bimolecular oxidation rate constant was measured in the range 1--200 mM using Tris--cacodylate or potassium phosphate buffers at pH 7.0, and electrodialyzed horse heart cytochrome c. The oxidation reaction proceeded very rapidly. Extrapolated to zero ionic strength, kox (approximately 9 X 10(9) M-1 S-1) was about 7% of that calculated for a diffusion-limited reaction. Since the exposed heme edge occupies only the order of 3% of the surface area, electron transfer apparently results at nearly every collision with the active-site region. An effective charge of + 7.8 units was estimated for the oxidation reaction. The rate of oxidation of Pseudomonas aeruginosa c551 was much slower (kox at mu = 0 was the order of 6 X 10(3)), and was not consistent with diffusion-limited kinetics.     

Superoxide dismutase mimetic activity of cytokinin-copper(II) complexes

Dissociation constants of cytokinins, derivatives of purine which form complexes with cupric ion, were determined by spectrophotometry and the stability constants of their copper complexes by pH titration. The values found for kinetin were 3.76, 9.96, 7.8, and 15.3 for pK1, pK2, logk1, and log beta 2, respectively, and those for 6-benzylaminopurine were, in the same order, 3.90, 9.84, 8.3, and 15.9. The copper(II) complexes with kinetin and 6-benzylaminopurine had superoxide dismutase mimetic activity, and the reaction rate constants with superoxide, which were determined by polarography, were 2.3 X 10(-7) M-1 s-1 for kinetin and 1.5 X 10(-7) M-1 s-1 for 6-benzylaminopurine at pH 9.8 and 25 degrees C.     

The reactivity of various free radicals with hyaluronic acid: steady-state and pulse radiolysis studies

The reactions of the primary water radicals with the biopolymer hyaluronic acid have been studied by pulse radiolysis. Bimolecular rate constants, expressed in terms of the disaccharide repeating sub-unit of hyaluronic acid, for OH., H. and eaq- were found to be 7 X 10(8) M-1 X s-1, 5 X 10(7) M-1 X s-1 and less than 5 X 10(6) M-1 X s-1, respectively. By comparing the viscosities of samples, gamma-irradiated in the steady state under a variety of conditions, with unirradiated controls, the efficiencies with which selected radicals cause chain breakage have been determined. Efficiencies of 30%, 15%, 0%, 0.2% and 5% were estimated for OH., H., eaq-, methanol radicals and tert-butanol radicals, respectively. The presence of oxygen during irradiation increased the extent of chain breakage by a factor of 1.75.     

Kinetics of the interaction of alpha-chymotrypsin with trypsin kallikrein inhibitor (Kunitz) in which the reactive-site peptide bond Lys-15--Ala-16 is split.

Modified trypsin kallikrein inhibitor (I*), with the reactive-site peptide bond Lys-15--Ala-16 split, reacts with alpha-chymotrypsin (E) via an intermediate X to the stable tetrahedral complex C:E + I in equilibrium X leads to C. Formation X constitutes a fast pre-equilibrium (equilibrium constant Kx = 7 X 10(-5) M, association rate constant kx = 4 X 10(3)M-1s-1) to the slow reaction X leads to C (rate constant kc = 2 X 10(-3) s-1), all values at pH 7.5. No intermediate X is observed when alpha-chymotrypsin reacts with I*-OMe in which the carboxyl group of Lys-15 is esterified by methanol. This observation as well as the different pH dependence of the overall association rate constants in the case of I* and I*-OMe indicate tha formation of X precedes formation of the acyl enzyme in the catalytic pathway. The data are compared to the similar results obtained with beta-trypsin and I* or I*-OMe.     

Chelated iron-catalyzed OH. formation from paraquat radicals and H2O2: mechanism of formate oxidation

Traces of iron, when complexed with either EDTA or diethylenetriaminepentaacetic acid (DTPA), catalyze an OH.-producing reaction between H2O2 and paraquat radical (PQ+.): H2O2 + PQ+.----PQ++ + OH. + OH-.[1]. Kinetic studies show that oxidation of formate induced by this reaction occurs by a Fenton-type mechanism, analagous to that assumed in the metal-catalyzed Haber-Weiss reaction, in which the rate determining step is H2O2 + Fe2+ (chelator)----Fe3+(chelator) + OH. + OH-,[7]; with k7 = 7 X 10(3) M-1 s-1 for EDTA and 8 X 10(2) M-1 s-1 for DTPA at pH 7.4. PQ+. rapidly reduces both Fe3+ (EDTA) and Fe3+ (DTPA), and hence allows both agents to catalyze [1] with comparable efficiency, in contrast to the much lower efficiency reported for the latter as a catalyst for the Haber-Weiss reaction. The catalytic properties of these chelating agents is attributed to their lowering of E0 (Fe3+/Fe2+) by 0.65 V, thus making [7] thermodynamically possible at pH 7. Approximately 2.5% of the OH. produced is consumed by internal or "cage" reactions, which decompose the chelator and produce CO2; however, the majority (97%) diffuses into the bulk solution and participates in competitive reactions with OH. scavengers.     

Kinetics of adrenodoxin reductase oxidation by non-physiologic electron acceptors

The reactions of NADPH oxidation by quinones and inorganic complexes catalyzed by NADPH: adrenodoxin reductase were studied. The catalytic constant for the enzyme at pH 7.0 is 20-25 s-1; the oxidative constants for the quinones vary from 5 X 10(5) to 1.1 X 10(3) M-1 s-1 and show an increase with a rise in the one-electron acceptor reduction potential. The mode of adrenodoxin reductase interaction with oxyquinones differs from that of the enzyme interaction with alkyl-substituted quinones and inorganic complexes. NADPH competitively inhibits electron acceptors, whereas NADP+ is a competitive inhibitor of NADPH and a uncompetitive inhibitor of electron acceptors. (Ki = 25 microM). The depth of FAD incorporation into the enzyme molecule as calculated according to the outer sphere electron transfer theory is 6.1 A.