Specificity of a Catabolic Pathway—a Lesson Learned from Indirect Assays

Studies with purified orcinol hydroxylase suggest that, contrary to previous conclusions, the enzymes of the orcinol pathway cannot transform analogous compounds to common metabolites. The substrate analogues of orcinol uncouple electron flow from reduced nicotinamide adenine dinucleotide to oxygen from the hydroxylation reaction catalyzed by orcinol hydroxylase.     

Affinity chromatography of Pseudomonas salicylate hydroxylase

In order to facilitate the purification of salicylate hydroxylase (salicylate 1-monooxygenase, EC 1.14.13.1) from Pseudomonas sp. RPP (ATCC 29351), an affinity chromatography procedure was developed employing immobilized salicylate as the affinity ligand. The immobilization was achieved by reacting p-aminosalicylate with the N-hydroxysuccinimide ester of Sepharose 4B-6-aminohexanoic acid. When the bacterial crude extract was chromatographed with this affinity column, salicylate hydroxylase was absorbed to the gel while the bulk of protein freely passed through. The absorbed enzyme was subsequently eluted from the affinity column by applying a 0–60 mm sodium salicylate gradient. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the enzymatically most active fraction of the affinity effluent revealed salicylate hydroxylase was by far the most predominant protein but there were also small amounts of contaminating proteins. However, a virtually homogeneous enzyme preparation was obtained when the crude extract was first fractionated with a DE-52 anion-exchange column followed by the affinity step. The enzyme preparation obtained by this two-step procedure showed a specific activity of 14.9 units/mg and an A₄₅₀:A₃₇₂:A₂₈₀ of 1.01:1:10.23. Because most of the enzymes belonging to the class of external flavoprotein monooxygenase utilize salicylate analogs as substrates and share many other common properties, there is a strong possibility that the salicylate column may be useful for the purification of other member monooxygenases.     

The stereochemistry of NADH utilization by the flavoenzyme monooxygenase orcinol hydroxylase.

Ribbons et al. (Ribbons, D.W., Ohta, Y., and Higgins, I.J. (1972) in Molecular Basis of Electron Transport, Miami Winter Symposic Series (Schultz, J., and Cameron, B.F., eds) Vol. 4, pp. 251-274, Academic Press, New York) presented a preliminary report that the flavoenzyme monooxygenase orcinol hydroxylase shows mixed type 4R, 4S stereospecificity with respect to dihydronicotinamide oxidation when resorcinol and m-cresol were used as substrate analogs. With the natural substrate orcinol, 4R chirality was maintained. In kinetic isotope experiments reported here, we demonstrate in fact that orcinol hydroxylase maintains 4R stereospecificity with respect to dihydronicotinamide oxidation with all three substrates, orcinol, resorcinol, and m-cresol. Deuterium and tritium kinetic isotope effects were detected under Vmax conditions with (4R)-[4-2H]-, and (4R)-[4-3H]NADH for all three substrates. No isotope effect was observed with (4S)-[4-2H]NADH and tritium labilization from assays with (4S)-[4-3H]-NADH was negligible in all cases.     

Nicotinamide Adenine Dinucleotide Phosphate-specific Isocitrate Dehydrogenase from a Higher Plant: Isolation and Characterization 1

Nicotinamide adenine dinucleotide phosphate-specific isocitrate dehydrogenase was extracted from etiolated pea (Pisum sativum L.) seedlings and was purified 65-fold. The purified enzyme exhibits one predominant protein band by polyacrylamide gel electrophoresis, which corresponds to the dehydrogenase activity as measured by the nitro blue tetrazolium technique. The reaction is readily reversible, the pH optima for the forward (nicotinamide adenine dinucleotide phosphate reduction) and reverse reactions being 8.4 and 6.0, respectively. The enzyme has different cofactor and inhibitor characteristics in the two directions. Manganese ions can be used as a cofactor for the reaction in each direction but magnesium ions only act as a cofactor in the forward reaction. Zinc ions, and to a lesser extent calcium ions, inhibit the enzyme at low concentrations when magnesium but not manganese is the metal activator. It is suggested that there is a fundamental difference between magnesium and manganese in the activation of the enzyme. The enzyme shows normal kinetics and the Michaelis contant for each substrate was determined. The inhibition by nucleotides, nucleosides, reaction products, and related compounds was studied. The enzyme shows a linear response to the mole fraction of reduced nicotinamide adenine dinucleotide phosphate when total nicotinamide adenine dinucleotide phosphate (nicotinamide adenine dinucleotide phosphate plus reduced nicotinamide adenine dinucleotide phosphate) is kept constant. Isocitrate in the presence of divalent metal ions will protect the enzyme from inactivation by p-chloromercuribenzoate. Protection is also afforded by manganese ions alone but not by magnesium ions alone There is a concerted inhibition of the enzyme by oxalacetate and glyoxylate.     

Further chemical characterization and biological activities of fluorescent I,N6-ethenoadenosine derivatives

The fluorescent 1,N⁶-ethenoadenosine derivatives of adenosine triphosphate, adenosine diphosphate, adenosine monophosphate, 3′:5′-cyclic adenosine monophosphate, adenosine and nicotinamide adenine dinucleotide have been prepared. Paper and thin layer chromatographic purification methods have been developed. Nuclear magnetic resonance and mass spectrum data indicate that only the purine ring has been modified.The 1,N⁶-ethenoadenosine triphosphate had about 70% of the activity of adenosine triphosphate as a substrate for total adenosine triphosphatase activity of hypophysectomized rat liver membranes. The 1,N⁶-ethenoadenosine diphosphate had about 86% of the activity of adenosine diphosphate as a substrate for adenosine diphosphatase of hypophysectomized rat liver membranes. The 1,N⁶-etheno derivative of nicotinamide adenine dinucleotide had about 8% of the activity of nicotinamide adenine dinucleotide as a substrate for nicotinamide adenine dinucleotide glycohydrolase and about 54% of the activity of nicotinamide adenine dinucleotide as a substrate for nicotinamide adenine dinucleotide pyrophosphatase of hypophysectomized rat liver membranes.K_m's for the ATPase, ADPase and yeast alcohol dehydrogenase using ε-ATP and ε-ADP and ε-NAD as substrates are presented.     

Role of molybdenum in nitrate reduction by chlorella

Molybdenum is absolutely required for the nitrate-reducing activity of the nicotinamide adenine dinucleotide nitrate reductase complex isolated from Chlorella fusca. The whole enzyme nicotinamide adenine dinucleotide nitrate reductase is formed by cells grown in the absence of added molybdate, but only its first activity (nicotinamide adenine dinucleotide diaphorase) is functional. The second activity of the complex, which subsequently participates also in the enzymatic transfer of electrons from nicotinamide adenine dinucleotide to nitrate (FNH₂-nitrate reductase), depends on the presence of molybdenum. Neither molybdate nor nitrate is required for nitrate reductase synthesis de novo, but ammonia acts as a nutritional repressor of the complete enzyme complex. Under conditions which exclude de novo synthesis of nitrate reductase, the addition of molybdate to molybdenum-deficient cells clearly increases the activity level of this enzyme, thus suggesting in vivo incorporation of the trace metal into the pre-existing inactive apoenzyme.     

Determination of the hydride transfer stereospecificity of nicotinamide adenine dinucleotide linked oxidoreductases by proton magnetic resonance.

A facile proton magnetic resonance technique is described for the determination of the coenzyme stereospecificity during hydride transfer reactions catalyzed by pyridine nucleotide dependent oxidoreductases. The reliability of this technique was demonstrated by examining the coenzyme stereospecificity of lactate, malate, and 3-phosphoglycerate dehydrogenases, which are known to be A-stereospecific enzymes, as well as triosephosphate and octopine dehydrogenases, which are known to be B-stereospecific enzymes. Furthermore, by applying this technique, it was shown that the previously unstudied enzymes D-beta-hydroxybutyrate and 4-aminobutanal dehydrogenases are B- and A-stereospecific enzymes, respectively. In addition, the nicotinamide adenine dinucleotide linked reaction of glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides was found to be B stereospecific, like the reaction of the nicotinamide adenine dinucleotide phosphate linked yeast enzyme.     

Glutamate dehydrogenase from Mycoplasma laidlawii

Mycoplasma laidlawii possesses a single glutamate dehydrogenase (GDH) with dual coenzyme specificity [specificity for nicotinamide adenine dinucleotide (H) and nicotinamide adenine dinucleotide phosphate (H)]. A purification procedure is reported which results in an enzyme preparation with a specific activity of 79.5 units/mg and which displays only one significant protein band after gel electrophoresis. This one band was determined, by activity staining, to have all of the GDH nucleotide specificities. The molecular weight of the enzyme is 250,000 +/- 10%, and it has a subunit size of about 48,000. The enzyme exhibits measurable activity with aspartate and pyruvate but is inactive with eight other possible substrates. Purine nucleotides do not affect the activity. The K(m) for reduced nicotinamide adenine dinucleotide was 1.8 x 10(-4)m. The optimal substrate concentrations and pH optimum for each of the respective GDH activities are also reported.     

Thiosulfate- and Sulfide-Dependent Pyridine Nucleotide Reduction and Gluconeogenesis in Intact Thiobacillus neapolitanus

Nicotinamide adenine dinucleotide phosphate (reduced form) is formed more rapidly after the addition of thiosulfate to suspensions of intact Thiobacillus neapolitanus in the absence of CO(2) than nicotinamide adenine dinucleotide (reduced form). Measurement of acid-stable metabolites shows this phenomenon to be the result of rapid reoxidation of nicotinamide adenine dinucleotide (reduced form) by 3-phosphoglyceric acid and other oxidized intermediates, which are converted to triose and hexose phosphates, and that, in reality, the rate of nicotinamide adenine dinucleotide (oxidized form) reduction exceeds that of nicotinamide adenine dinucleotide phosphate (oxidized form) by approximately 4.5-fold. The overall rate of pyridine nucleotide reduction by thiosulfate (264 nmol per min per mg of protein) is in excess of that rate needed to sustain growth. Pyridine nucleotide reduction, adenosine triphosphate synthesis, and carbohydrate synthesis are prevented by the uncoupler m-Cl-Carbonylcyanide phenylhydrazone. Sodium amytal inhibits pyridine nucleotide reduction and carbohydrate synthesis are prevented by the uncoupler m-Cl-carbonylcyanide observations are reproduced when sulfide serves as the substrate. The rate of pyridine nucleotide anaerobic reduction with endogenous substrates or thiosulfate is less than 1% of the aerobic rate with thiosulfate. We conclude that the principal, if not the only, pathway of pyridine nucleotide reduction proceeds through an energy-dependent and amytal-sensitive step when either thiosulfate or sulfide is used as the substrate.     

Alpha-glycerophosphate oxidase in Streptococcus faecium F 24

alpha-Glycerophosphate oxidase, in a strain of Streptococcus faecium, was found to be adaptive to aerated conditions of growth. The enzyme was purified and found to mediate electron transfer from alpha-glycerophosphate to O(2), with the production of stoichiometric concentrations of H(2)O(2) and dihydroxyacetone phosphate. The enzyme is an anionic flavoprotein, with flavine adenine dinucleotide as the apparent prosthetic group. By manometric methods, a K(m) of 6 x 10(-3)m, with reference to substrate concentration, was obtained. An active reduced nicotinamide adenine dinucleotide diaphorase was closely associated with this enzyme in chromatographic mobility on ECTEOLA-cellulose. The purified alpha-glycerophosphate oxidase was not inhibited by KCN, azide, or sulfhydryl reagents, nor was it stimulated by alpha-lipoate, yeast extract, or other supplements.     

A Metaphosphate-dependent Nicotinamide Adenine Dinucleotide Kinase from Brevibacterium ammoniagenes

The enzyme utilizing metaphosphate for nicotinamide adenine dinucleotide phosphorylation was purified 500-fold from B. ammoniagenes and its properties were studied. The isolated enzyme appeared homogeneous on disc gel electrophoresis; its molecular weight was determined to be 9.0 × 10⁴ by gel filtration. This enzyme specifically phosphorylated nicotinamide adenine dinucleotide at the optimum pH at 6.0. Of phosphoryl donors tested, metaphosphate was most effective for the reaction, and adenosine-5′-triphosphate was less effective. The activity was inhibited by adenosine-5′-monophosphate, adenosine-5′-diphosphate or reduced pyridine nucleotides. The enzyme did not exhibit catalytic activity in the absence of a divalent cation. We concluded that the enzyme phosphorylating nicotinamide adenine dinucleotide in the presence of metaphosphate is distinct from adenosine-5′-triphosphate-dependent nicotinamide adenine dinucleotide kinase, and tentatively designated it metaphosphate-dependent nicotinamide adenine dinucleotide kinase.     

Cytochrome p-450-dependent hydroxylation of lauric Acid at the subterminal position and oxidation of unsaturated analogs in wheat microsomes

Microsomes from etiolated wheat (Triticum aestivum L. cv Etoile de Choisy) shoots catalyzed the reduced nicotinamide adenine dinucleotide phosphate-dependent hydroxylation of lauric acid predominantly at the subterminal or (ω-1) position (65%). Minor amounts of 10-hydroxy- (31%) and 9-hydroxylaurate (4%) were also formed. The reaction was catalyzed by cytochrome P-450, since enzyme activity was strongly inhibited by tetcyclacis, carbon monoxide, and antibodies against NADPH-cytochrome c (P-450)-reductase. The apparent K_m for lauric acid was estimated to be 8.5 ± 2.0 μm. Seed treatment with the safener naphthalic acid anhydride or treatment of seedlings with phenobarbital increased cytochrome P-450 content and lauric acid hydroxylase (LAH) activity of the microsomes. A combination of both treatments further stimulated LAH activity. A series of radiolabeled unsaturated lauric acid analogs (8-, 9-, 10-, and 11-dodecenoic acids) was used to explore the regioselectivity and catalytic capabilities of induced wheat microsomes. It has been found that wheat microsomes catalyzed the reduced nicotinamide adenine dinucleotide phosphate-dependent epoxidation of sp2 carbons concurrently with hydroxylation at saturated positions. The regioselectivity of oxidation of the unsaturated substrates and that of lauric acid were similar. Preincubation of wheat microsomes with reduced nicotinamide adenine dinucleotide phosphate and 11-dodecenoic acid resulted in a partial loss of LAH activity.     

A new ternary complex between horse liver alcohol dehydrogenase, NAD+, and acetone

Acetone was found to form a dead-end ternary complex with horse liver alcohol dehydrogenase and oxidized nicotinamide adenine dinucleotide (NAD⁺) when the reactants were incubated for a long time at relatively high concentrations. The complex formation was demonstrated by measuring the increase in absorbance at 320 nm, the quenching of protein fluorescence, and the loss of enzyme activity. Since acetone is a substrate of liver alcohol dehydrogenase, and the presence of acetaldehyde or pyrazole prevents acetone from forming the dead-end complex with liver alcohol dehydrogenase and NAD⁺, the acetone molecule in the complex may be bound to the substrate binding site of liver alcohol dehydrogenase. The dissociation of the complex was demonstrated by prolonged dialysis or by addition of reduced nicotinamide adenine dinucleotide (NADH) and iso-butyramide. A modified nicotinamide adenine dinucleotide was obtained as a main product from the dead-end complex after dissociation of the complex or denaturation of the apoenzyme. The modified nicotinamide adenine dinucleotide was found to exhibit an absorption spectrum similar to that of NADH; however, it was not oxidizable by liver alcohol dehydrogenase in the presence of acetaldehyde and exhibited no fluorescence.     

Expanding the biocatalytic toolbox of flavoprotein monooxygenases from Rhodococcus jostii RHA1

With the aim to enlarge the set of available flavoprotein monooxygenases, we have cloned 8 unexplored genes from Rhodococcus jostii RHA1 that were predicted to encode class B flavoprotein monooxygenases. Each monooxygenase can be expressed as soluble protein and has been tested for conversion of sulfides and ketones. Not only enantioselective sulfoxidations, but also enantioselective Baeyer–Villiger oxidations could be performed with this set of monooxygenases. Interestingly, in contrast to known class B flavoprotein monooxygenases, all studied biocatalysts showed no nicotinamide coenzyme preference. This feature coincides with the fact that the respective sequences appear to form a discrete group of sequence related proteins, distinct from the known class B flavoprotein monooxygenases subclasses: the so-called flavin-containing monooxygenases (FMOs), N-hydroxylating monooxygenases (NMOs) and Type I Baeyer–Villiger monooxygenases (BVMOs). Taken together, these data reveal the existence of a new subclass of class B flavoprotein monooxygenases, which we coined as Type II FMOs, that can perform Baeyer–Villiger oxidations and accept both NADPH and NADH as coenzyme. The uncovered biocatalytic properties of the studied Type II FMOs make this newly recognized subclass of monooxygenases of potential interest for biocatalytic applications.     

Biochemical characterization of a flavin adenine dinucleotide-dependent monooxygenase, ornithine hydroxylase from Pseudomonas aeruginosa, suggests a novel reaction mechanism

Pyoverdin is the hydroxamate siderophore produced by the opportunistic pathogen Pseudomonas aeruginosa under the iron-limiting conditions of the human host. This siderophore includes derivatives of ornithine in the peptide backbone that serve as iron chelators. PvdA is the ornithine hydroxylase, which performs the first enzymatic step in preparation of these derivatives. PvdA requires both flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide phosphate (NADPH) for activity; it was found to be a soluble monomer most active at pH 8.0. The enzyme demonstrated Michaelis-Menten kinetics in an NADPH oxidation assay, but a hydroxylation assay indicated substrate inhibition at high ornithine concentration. PvdA is highly specific for both substrate and coenzyme, and lysine was shown to be a nonsubstrate effector and mixed inhibitor of the enzyme with respect to ornithine. Chloride is a mixed inhibitor of PvdA with respect to ornithine but a competitive inhibitor with respect to NADPH, and a bulky mercurial compound (p-chloromercuribenzoate) is a mixed inhibitor with respect to ornithine. Steady-state experiments indicate that PvdA/FAD forms a ternary complex with NADPH and ornithine for catalysis. PvdA in the absence of ornithine shows slow substrate-independent flavin reduction by NADPH. Biochemical comparison of PvdA to p-hydroxybenzoate hydroxylase (PHBH, from Pseudomonas fluorescens) and flavin-containing monooxygenases (FMOs, from Schizosaccharomyces pombe and hog liver microsomes) leads to the hypothesis that PvdA catalysis proceeds by a novel reaction mechanism.     

Redox involvement in acid secretion in the amphibian gastric mucosa

Summary Gastric fundic metabolism was studied by spectroscopic observation in frog mucosa during transitions of secretory status in vitro and by direct measurement of pyridine nucleotides and associated metabolites in biopsies of dog fundic mucosa also during secretory oxidation of the redox components from flavin adenine dinucleotide (FAD) to cytochromea ₃. Addition of histamine resulted in reduction of these components with onset of secretion by about 50%. In contrast, the effect of apparently, burimamide and subsequently histamine on the ratio of nicotinamide adenine dinucleotide to nicotinamide adenine dinucleotide, reduced (NAD⁺/NADH) was relatively slight. Further, the presence of burimamide substantially reduces the effect of amytal on the pyridine nucleotide spectrum and abolishes the effect of amytal on FAD and the cytochromes. Measurements of lactate, pyruvate, -ketoglutarate, NH₃ and glutamate in the dog showed that whereas the calculated NAD⁺/NADH ratio in the cytoplasm declined with onset of secretion, the calculated mitochondrial ratio rose. No change was noted in the nicotinamide adenine dinucleotide phosphate/nicotinamide adenine dinucleotide phosphate, reduced (NADP⁺/NADPH) ratio. It is concluded that (1) H₂ antagonists act by blocking substrate flow into the mitochondrial respiratory chain, (2) conversely, histamine stimulation acts at the level of substrate mobilization, and (3) there may be a cross-over in the mitochondrial chain between NAD⁺ and FAD.     

Metabolism of resorcinylic compounds by bacteria: alternative pathways for resorcinol catabolism in Pseudomonas putida.

Two strains of Pseudomonas putida isolated by enrichment cultures with orcinol as the sole source of carbon were both found to grow with resorcinol. Data are presented which show that one strain (ORC) catabolizes resorcinol by a metabolic pathway, genetically and mechanistically distinct from the orcinol pathway, via hydroxyquinol and ortho oxygenative cleavage to give maleylacetate, but that the other strain (O1) yields mutants that utilize resorcinol. One mutant strain, designated O1OC, was shown to be constitutive for the enzymes of the orcinol pathway. After growth of this strain on resorcinol, two enzymes of the resorcinol pathway are also induced, namely hydroxyquinol 1,2-oxygenase and maleylacetate reductase. Thus hydroxyquniol, formed from resorcinol, undergoes both ortho and meta diol cleavage reactions with the subsequent formation of both pyruvate and maleylacetate. Evidence was not obtained for the expression of resorcinol hydroxylase in strain O1OC; the activity of orcinol hydroxylase appears to be recruited for this hydroxylation reaction. P. putida ORC, on the other hand, possesses individual hydroxylases for orcinol and resorcinol, which are specifically induced by growth on their respective substrates. The spectral changes associated with the enzymic and nonenzymic oxidation of hydroxyquinol are described. Maleylacetate was identified as the product of hydroxyquinol oxidation by partially purified extracts obtained from P. putida ORC grown with resorcinol. Its further metabolism was reduced nicotinamide adenine dinucleotide dependent.     

Hydrogenase Activity and the H2-Fumarate Electron Transport System in Bacteroides fragilis

Hydrogenase activity and the H₂-fumarate electron transport system in a carbohydrate-fermenting obligate anaerobe, Bacteroides fragilis, were investigated. In both whole cells and cell extracts, hydrogenase activity was demonstrated with methylene blue, benzyl viologen, flavin mononucleotide, or flavin adenine dinucleotide as the electron acceptor. A catalytic quantity of benzyl viologen or ferredoxin from Clostridium pasteurianum was required to reduce nicotinamide adenine dinucleotide or nicotinamide adenine dinucleotide phosphate with H₂. Much of the hydrogenase activity appeared to be associated with the soluble fraction of the cell. Fumarate reduction to succinate by H₂ was demonstrable in cell extracts only in the presence of a catalytic quantity of benzyl viologen, flavin mononucleotide, flavin adenine dinucleotide, or ferredoxin from C. pasteurianum. Sulfhydryl compounds were not required for fumarate reduction by H₂, but mercaptoethanol and dithiothreitol appeared to stimulate this activity by 59 and 61%, respectively. Inhibition of fumarate reduction by acriflavin, rotenone, 2-heptyl-4-hydroxyquinoline-N-oxide, and antimycin A suggest the involvement of a flavoprotein, a quinone, and cytochrome b in the reduction of fumarate to succinate. The involvement of a quinone in fumarate reduction is also apparent from the inhibition of fumarate reduction by H₂ when cell extracts were irradiated with ultraviolet light. Based on the evidence obtained, a possible scheme for the flow of electrons from H₂ to fumarate in B. fragilis is proposed.     

Bilevel disulfide group reduction in the activation of c(4) leaf nicotinamide adenine dinucleotide phosphate-malate dehydrogenase

The time course of thioredoxin-mediated reductive activation of isolated Zea mays nicotinamide adenine dinucleotide phosphatemalate dehydrogenase is highly sigmoidal in nature. We examined the factors affecting these kinetics, including the thiol-disulfide status of unactivated and activated forms of the enzyme. The maximum steady rate of activation was increased, and the length of the lag in activation decreased, as the concentrations of thioredoxin-m, dithiothreitol, and KCl were increased. The lag in activation (sigmoidicity) was eliminated by preincubating the unactivated enzyme with 100 mm 2-mercaptoethanol; this pretreatment did not activate the enzyme. Unactivated nicotinamide adenine dinucleotide phosphate-malate dehydrogenase was found to contain approximately two SH groups per subunit, increasing to about four SH per subunit after pretreatment with 2-mercaptoethanol and six SH per subunit after activation by incubating the enzyme with dithiothreitol. We suggest that reduction of one particular higher redox potential disulfide group in unactivated nicotinamide adenine dinucleotide phosphate-malate dehydrogenase facilitates the subsequent reduction of the critical S-S group (regulatory S-S) necessary to generate the active form of the enzyme.     

Interactions between spinach ferredoxin and other electron carriers. The involvement of a ferredoxin:cytochrome c complex in the ferredoxin-linked cytochrome c reductase activity of ferredoxin:NADP+ oxidoreductase.

The trinitrophenylation of a single amino group of spinach ferredoxin abolishes its ability to inhibit the diaphorase activity of the flavoprotein, ferredoxin:NADP oxidoreductase (EC 1.6.7.1); in contrast, the ability of ferredoxin to participate in the ferredoxin-linked cytochrome c reductase activity catalyzed by the flavoprotein is unaffected. Comparison with previously published results [Davis, D. J., and San Pietro, A. (1977) Biochem. Biophys. Res. Commun.74, 33–40]indicates that the site of interaction between ferredoxin and the flavoprotein resulting in inhibition if diaphorase activity is responsible for the spectrally observable 1:1 complex between the two proteins and is identical to the site of ferredoxin involvement in NADP photoreduction. The role of ferredoxin in the ferredoxin-linked cytochrome c reductase activity of the flavoprotein has been reexamined under conditions were the entire electron-accepting system (rather than just the ferredoxin component) is rate limiting. The data support a mechanism by which ferredoxin can bind either to the flavoprotein or to cytochrome c, and the ferredoxin:cytochrome c complex serves as the true substrate for reduction by the flavoprotein. Furthermore, Chromatographic evidence is presented for the formation of complexes between ferredoxin and cytochrome c.