Interaction and identification of ubiquinone-binding proteins in ubiquinol-cytochrome c reductase by azido-ubiquinone derivatives

Various azido-ubiquinone derivatives were synthesized and characterized. 3-Azido-2-methyl-5-methoxy-6-(3,7-dimethyloctyl)-1,4-benzoquinone was found to be suitable for the study of specific interaction between ubiquinone (Q) and protein. It was synthesized with high specific radioactivity and used to identify the Q-binding proteins in purified ubiquinol-cytochrome c reductase. This azido-Q derivative showed partial efficiency in restoring activity to the Q- and phospholipids-depleted ubiquinol-cytochrome c reductase in the absence of light. Azido-Q derivative treated samples, however, became completely inactivated upon photolysis, and the inactivation was not reversed by addition of Q derivatives. The redox state of the azido-Q derivative has little effect on the Q-binding affinity. Two protein subunits with Mr = 37,000 and 17,000 were found to be heavily labeled when depleted ubiquinol-cytochrome c reductase was treated with [3H] azido-Q derivative followed by photolysis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The amount of radioactive labeling of the Mr = 17,000 protein was proportional to the degree of inactivation and affected by the presence of phospholipids. The radioactive labeling of the Mr = 37,000 protein subunit, however, showed no correlation with degree of inactivation and was not affected by phospholipids. Since the radiolabeling at the Mr = 17,000 protein subunit was affected by phospholipids and correlated with the enzymatic activity, this subunit is probably the Q-binding protein in this enzyme complex (QPc). The inhibition of enzymatic activity by n-heptyl-4-hydroxyquinoline-N-oxide was easily reversed by addition of the azido-Q derivative. The distribution of radioactivity among the subunits of ubiquinol-cytochrome c reductase was not affected by the presence of antimycin A, 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole or n-heptyl-4-hydroxyquinoline-N-oxide, suggesting that the binding site(s) of these inhibitors are not the Q-binding site.     

The catalytic role of subunit IV of the cytochrome b6-f complex from spinach chloroplast

The catalytic role of subunit IV, the Mr 17,000 protein, in the chloroplast cytochrome b6-f complex was established through trypsinolysis of the complex under controlled conditions. When purified chloroplast cytochrome b6-f complex, 1 mg/ml, in 50 mM Tris-succinate buffer (pH 7.0) containing 1% sodium cholate and 10% glycerol is treated with 80 micrograms of trypsin at room temperature for various lengths of time, the activity of the cytochrome b6-f complex decreases as the incubation time increases. A maximal inactivation of 80% is reached at 7 min of incubation. The trypsin inactivation is accompanied by the destruction of the proton translocation activity of the complex. No alteration of absorption and EPR spectral properties was observed in the trypsin-inactivated complex. Subunit IV is the only subunit in the cytochrome b6-f complex that is digested by trypsin, and the degree of digestion correlates with the decrease of electron transfer activity. The binding of azido-Q to subunit IV of the complex decreases as the extent of inactivation of the cytochrome b6-f complex by trypsin increases. The residue molecular mass of trypsin cleaved subunit IV is about 14 kDa, suggesting that the cleavage site is at lysine 119 or arginine 125 or 126. When the thylakoid membrane was assayed for cytochrome b6-f complex activity, very little activity was observed; and the activity was not sensitive to trypsinolysis. Upon sonication, activity and sensitivity to trypsinolysis was greatly increased, suggesting that subunit IV protrudes from the lumen side of the membrane.     

The Rhodospirillum rubrum cytochrome bc1 complex: peptide composition, prosthetic group content and quinone binding

A cytochrome bc1 complex, essentially free of bacteriochlorophyll, has been purified from the photosynthetic purple non-sulfur bacterium Rhodospirillum rubrum. The complex catalyzes electron flow from quinol to cytochrome c (turnover number = 75 s-1) that is inhibited by low concentrations of antimycin A and myxothiazol. The complex contains only three peptide subunits: cytochrome b (Mr = 35,000); cytochrome c1 (Mr = 31,000) and the Rieske iron-sulfur protein (Mr = 22,400). Em values (pH 7.4) were measured for cytochrome c1 (+320 mV) and the two hemes of cytochrome b (-33 and -90 mV). Electron flow from quinol to cytochrome c is inhibited when the complex is pre-illuminated in the presence of a ubiquinone photoaffinity analog (azido-Q). During illumination, the azido-Q becomes covalently attached to the cytochrome b peptide and, to a lesser extent, to cytochrome c1.     

Use of an azido-ubiquinone derivative to identify subunit I as the ubiquinol binding site of the cytochrome d terminal oxidase complex of Escherichia coli

The radiolabeled, photoreactive azido-ubiquinone derivative (azido-Q), 3-azido-2-methyl-5-methoxy-6-(3,7-dimethyl-[3H]octyl)- 1,4-benzoquinone, was used to investigate the active site of ubiquinol oxidase activity of the cytochrome d complex, a two-subunit terminal oxidase of Escherichia coli. The azido-Q, when reduced by dithioerythritol, was shown to support enzymatic oxygen consumption by the cytochrome d complex that was 8% of the rate observed with ubiquinol-1. This observation provided the rationale behind further studies of the possible photoinactivation and labeling of the active site by this azido-Q. Ten min of photolysis of the purified cytochrome d complex in the presence of the azido-Q resulted in a 60% loss of the ubiquinol-1 oxidase activity. Uptake of the radiolabeled azido-Q by the cytochrome d complex was correlated to the photoinactivation of the ubiquinol-1 oxidase activity. Both increased linearly during the first 4 min of photolysis and reached 90% of the maximum within 10 min. Photolysis times longer than 10 min resulted in no increase in the maximum of 2 mol of azido-Q incorporated per mol of enzyme. The rate of azido-Q uptake by subunit I, but not subunit II, correlated well with the rate of loss of ubiquinol oxidase activity. Use of ubiquinol-0, which is not oxidized by the enzyme, to competitively inhibit radiolabeling of nonspecific binding sites, resulted in a significant decrease (42%) of azido-Q labeling of subunit II while it did not affect the labeling of subunit I. After photolysis for 4 min, the ratio of radiolabeled azido-Q in subunits I to II of the complex was 4.3 to 1.0. These observations support the conclusion that the ubiquinol substrate binding site is located on subunit I of the cytochrome d complex.     

Identification of the ubiquinone-binding domain in the disulfide catalyst disulfide bond protein B.

Disulfide bond (Dsb) formation is catalyzed in the periplasm of prokaryotes by the Dsb proteins. DsbB, a key enzyme in this process, generates disulfides de novo by using the oxidizing power of quinones. To explore the mechanism of this newly described enzymatic activity, we decided to study the ubiquinone-protein interaction and identify the ubiquinone-binding domain in DsbB by cross-linking to photoactivatable quinone analogues. When purified Escherichia coli DsbB was incubated with an azidoubiquinone derivative, 3-azido-2-methyl-5-[(3)H]methoxy-6-decyl-1,4-benzoquinone ([(3)H]azido-Q), and illuminated with long wavelength UV light, the decrease in enzymatic activity correlated with the amount of 3-azido-2-methyl-5-methoxy-6-decyl-1,4-benzoquinone (azido-Q) incorporated into the protein. One azido-Q-linked peptide with a retention time of 33.5 min was obtained by high performance liquid chromatography of the V8 digest of [(3)H]azido-Q-labeled DsbB. This peptide has a partial NH(2)-terminal amino acid sequence of NH(2)-HTMLQLY corresponding to residues 91-97. This sequence occurs in the second periplasmic domain of the inner membrane protein DsbB in a loop connecting transmembrane helices 3 and 4. We propose that the quinone-binding site is within or very near to this sequence.     

Herbicide and plastoquinone-binding proteins of Photosystem II reaction center complexes from the thermophilic cyanobacterium,Synechococcus sp.

Photoaffinity labeling of Synechococcus Photosystem (PS) II preparations with radioactive azido-derivatives of three herbicides and of plastoquinone was carried out to identify herbicide and plastoquinone-binding proteins. [¹⁴C]Azido-atrazine and [¹⁴C]azido-monuron specifically labeled the 28 kDa polypeptide of the PS II reaction center complex, which is sensitive to 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea (DCMU). No specific labeling of this polypeptide with azido-atrazine was found in CP2-b (PS II reaction center lacking the 40 kDa subunit) which is insensitive to DCMU. [³H]Azido-dinoseb reacted with the 28 kDa polypeptide and the 47 kDa chlorophyll-carrying protein. The labeling with [³H]azido-plastoquinone resulted in the incorporation of the radioactivity exclusively into the 47 kDa polypeptide. It is concluded that the 28 kDa polypeptide is the herbicide-binding protein of the cyanobacterium and that the 47 kDa polypeptide has a binding site for plastoquinone and for phenol-type herbicides.     

The ubiquinone-binding site in NADH:ubiquinone oxidoreductase from Escherichia coli

An azido-ubiquinone derivative, 3-azido-2-methyl-5-methoxy[3H]-6-decyl-1,4-benzoquinone ([3H]azido-Q), was used to study the ubiquinone/protein interaction and to identify the ubiquinone-binding site in Escherichia coli NADH:ubiquinone oxidoreductase (complex I). The purified complex I showed no loss of activity after incubation with a 20-fold molar excess of [3H]azido-Q in the dark. Illumination of the incubated sample with long wavelength UV light for 10 min at 0 degrees C caused a 40% decrease of NADH:ubiquinone oxidoreductase activity. SDS-PAGE of the complex labeled with [3H]azido-Q followed by analysis of the radioactivity distribution among the subunits revealed that subunit NuoM was heavily labeled, suggesting that this protein houses the Q-binding site. When the [3H]azido-Q-labeled NuoM was purified from the labeled reductase by means of preparative SDS-PAGE, a 3-azido-2-methyl-5-methoxy-6-decyl-1,4-benzoquinone-linked peptide, with a retention time of 41.4 min, was obtained by high performance liquid chromatography of the protease K digest of the labeled subunit. This peptide had a partial NH2-terminal amino acid sequence of NH2-VMLIAILALV-, which corresponds to amino acid residues 184-193 of NuoM. The secondary structure prediction of NuoM using the Toppred hydropathy analysis showed that the Q-binding peptide overlaps with a proposed Q-binding motif located in the middle of the transmembrane helix 5 toward the cytoplasmic side of the membrane. Using the PHDhtm hydropathy plot, the labeled peptide is located in the transmembrane helix 4 toward the periplasmic side of the membrane.     

Quinone interactions with the chloroplast cytochrome b6-f complex

The requirements for reconstitution of electron transfer activity with a plastoquinone (PQ)-depleted cytochrome b6-f complex from spinach have been considered. Full restoration of activity measured as plastocyanin reduction with either duroquinol in the dark or Photosystem II (PSII) in the light requires both PQ-9 and phospholipid. However, a substantial dark activity can be observed with duroquinol and phospholipid in the absence of any added PQ-9. PSII, with its associated PQ molecules, can also donate electrons in the light to the cytochrome complex which has been depleted of plastoquinone. Electron donation by duroquinol in the dark to the PQ-depleted cytochrome complex is stimulated by PSII, and this stimulation is dependent on the presence of the two PQ molecules in the PSII preparation. Measurements of proton translocation with the PQ-depleted complex indicate this quinone is not required for the observed H+/e- ratio of 2. Studies of cytochrome b6 kinetics with the free and liposome-incorporated PQ-depleted complex show this cytochrome undergoes redox reactions similar to those of a control complex which contains PQ. These results indicate the PQ that copurifies with the cytochrome complex is not essential for any of the measured activities. These findings are considered in relation to a quinone binding site(s) in the cytochrome complex which is not specific to PQ but can bind other quinones, such as duroquinol, in a lipid-dependent process.     

Light inhibition of respiration is due to a dual function of the cytochrome b6-f complex and the plastocyanin/cytochrome c-553 pool in Aphanocapsa

Studies of the respiratory electron transport pathway in the blue-green alga, Aphanocapsa, demonstrated the presence of cytochrome oxidase and a cytochrome complex. The use of antimycin A showed only the occurrence of a plastidal type of cytochrome complex (the cytochrome b6-f complex), which is insensitive to this inhibitor. Determination of the extent of photooxidation of cytochromes c-553 and f-556 under conditions of high and low cytochrome oxidase activities indicated an electron flow through both cytochromes to cytochrome oxidase. Direct evidence for a common segment of photosynthetic and respiratory electron transport from plastoquinone via the cytochrome b6-f complex to the soluble plastocyanin/cytochrome c-553 pool, as well as a competition between cytochrome oxidase and Photosystem I for reductants in this pool in the light, was obtained by measurements of electron transport with suitable electron donors in this alga.     

Preparation and reconstitution of a phospholipid deficient cytochrome b6-f complex from spinach chloroplasts

A simple, rapid procedure suitable for large scale preparation of a lipid deficient cytochrome b6-f complex from spinach chloroplasts has been developed. The procedure involves solubilization with a mixture of sodium cholate and octylglucoside, ammonium sulfate fractionation and calcium phosphate column chromatography. The purified complex contains, in nanomoles per milligram protein, 20.6 cytochrome b, 10.8 cytochrome f and 54 phospholipids. The purified complex has little plastoquinol-cytochrome c reductase activity in the absence of added lipid. Full reductase activity was reconstituted by the addition of plastoquinone prior to the addition of lipid.     

The cytochrome bc) complex of yeast mitochondria. Site of translation of the polypeptides in vivo.

1.Yeast cells were labelled with radioactive amino acids in the presence of cycloheximide and the cytochrome bc1 complex was isolated from them as described in the preceding paper (Katan, M.B.., Pool, L. & Groot, G.S.P. (1976)Eur. J. Biochem, 65, 95-105). After analysis of this preparation by sodium dodecylsulphate polyacrylamide gel electrophoresis only one band, with an apparent Mr of 32000, was found to have incorporated radioactivity. The amount of label in the band was low, but could be increased approximately 5-fold by preincubating the cells in erythromycin before the labelling period. 2. Cells were labelled in the presence of chloramphenicol and the cytochrome bc1 complex was isolated by (NH4)2SO4 fractionation. Upon electrophoresis in the presence of sodium dodecylsulphate only four of the six bands that belong to the complex were found to have incorporated radioactivity; no radioactivity was found in the bands with an Mr of 40000 and 17000. The same result was obtained after labelling in the presence of acriflavin. If, however, the cytochrome bc1 complex was isolated by immunoprecipitation, all bands were found to have incorporated radioactivity in the presence of chloramphenicol. The amount of radioactivity in the Mr 32000 band was now clearly depressed. 3. It is concluded that of the seven polypeptides of the cytochrome bc1 complex of yeast only one is made on mitochondrial ribosomes. This polypeptide has an Mr of 32000 and is probably associated with cytochrome b.     

Chloroplast protein phosphorylation and chlorophyll fluorescence quenching. Activation by tetramethyl-p-hydroquinone, an electron donor to plastoquinone

When tetramethyl-p-benzoquinone (TMQ) is reduced to tetramethyl-p-hydroquinone (TMQH₂) by NaBH₄, TMQH₂ will act as an electron donor in isolated chloroplasts. The resulting electron transport is highly sensitive to inhibition by 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB), and the site of donation is inferred to be plastoquinone, in agreement with previous findings. In contrast, when TMQ is added to chloroplasts with ascorbate as reductant, the resulting electron transport is relatively insensitive to DBMIB, and so plastoquinone is assumed not to be involved. In darkness, TMQH₂ activates the chloroplast protein kinase that phosphorylates the light-harvesting chlorophyll a/b-protein complex (LHCP), while TMQ with ascorbate does not. TMQH₂ also activates ATP-dependent chlorophyll fluorescence quenching to a much greater extent than does TMQ with ascorbate. These findings are explained by the recent proposal that phosphorylation of LHCP is activated by reduced plastoquinone. They are therefore evidence for plastoquinone-regulated protein phosphorylation as a mechanism for self-adjustment of distribution of excitation between the two light reactions of photosynthesis.     

A ubiquinone derivative that inhibits mitochondrial cytochrome b-c1 complex but not chloroplast cytochrome b6-f complex activity

A ubiquinone derivative, 3-chloro-5-hydroxyl-2-methyl-6-decyl- 1,4-benzoquinone (3-CHMDB), which shows different effects on the mitochondrial cytochrome b-c1 complex and chloroplast cytochrome b6-f complex, has been synthesized and characterized. When the cytochrome b-c1 complex is treated with varying concentrations of 3-CHMDB and assayed at constant substrate (Q2H2) concentration, a 50% inhibition is observed when 2 mol of 3-CHMDB per mol of enzyme are used. The degree of inhibition is dependent on the substrate concentration. When ubiquinol-cytochrome c reductase is treated with 2 mol of 3-CHMDB per mol of enzyme, less inhibition is observed with a lower substrate concentration, suggesting the possible existence of two forms of reductases: one with a high affinity for ubiquinone and another with a low affinity. 2-Chloro-5-hydroxyl-3-methyl-6-decyl-1,4-benzoquinone (2-CHMDB), an isomer of 3-CHMDB, shows much less inhibition of the mitochondrial cytochrome b-c1 complex, suggesting that the quinone binding site in this complex is highly specific. In contrast to the inhibition observed with the cytochrome b-c1 complex, 3-CHMDB causes no inhibition of the plastoquinol-plastocyanin reductase activity of chloroplast cytochrome b6-f complex, regardless of whether plastoquinol-2 or ubiquinol-2 is used as substrate. 3-CHMDB restores the dibromothymoquinone-altered EPR spectra of iron-sulfur protein in both complexes. In the case of the cytochrome b6-f complex, 3-CHMDB also partially restores the dibromothymoquinone-inhibited activity. Reduced form 3- or 2-CHMDB is oxidizable by the cytochrome b6-f complex, but not by the cytochrome b-c1 complex. These results suggest that the quinol oxidizing sites in the cytochrome b6-f complex may differ from those in the mitochondrial cytochrome b-c1 complex.     

Some properties of Escherichia coli glutamine synthetase after limited proteolysis by subtilisin.

Escherichia coli glutamine synthetase is inactivated by subtilisin. Protection against inactivation is afforded by glutamine and ammonium ions. One large fragment (Mr = 35,000) is identified by sodium dodecyl sulfate-gel electrophoresis and carries adenylylation site. Smaller quantities of two other fragments (Mr = 17,000 and 15,000, respectively) are als observed oo observed on the gel. tthe nicked protein remains dodecameric, as evidenced by electrophoresis and centrifugation. It has retained the binding properties toward ADP and Ci-bacron blue and undergoes conformation changes upon binding, as does the intact protein. It is recognized by the antiserum raised against the native enzyme. The nicked protein also remains an excellent substrate of E. coli adenylyltransferase.     

The human ubiquitin carrier protein E2(Mr = 17,000) is homologous to the yeast DNA repair gene RAD6.

Components of the ubiquitin conjugating system were purified from human placenta by covalent affinity chromatography on ubiquitin sepharose. In contrast to E2 preparations obtained from rabbit reticulocytes and erythrocytes or Saccharomyces cerevisiae, the placental E2 preparation lacks E2(Mr = 14,000) and E2(Mr = 20,000) which are both unique in catalysing the ligase-independent transfer of ubiquitin to histones. A novel technique was employed to detect ubiquitin carrier function of the E2 proteins after SDS-electrophoresis and blotting to nitrocellulose. A cDNA of E2(Mr = 17,000) was isolated from a human cDNA library by screening with a degenerate oligonucleotide whose sequence was based on a partial amino acid sequence obtained from an E2(Mr = 17,000) peptide. Sequence analysis demonstrated an identity of 69% in the primary sequence of human E2(Mr = 17,000) and the protein encoded by the yeast DNA repair gene RAD6, which was recently shown to be an E2 species in yeast. Such a high degree of similarity between the human E2(Mr = 17,000) and the yeast DNA repair enzyme is suggestive of important common structural constraints or roles in addition to ubiquitin carrier activity, since in yeast this function itself is not necessarily dependent on high conservation of primary structure.     

The role of the supernumerary subunit of Rhodobacter sphaeroides cytochrome bc1 complex

The smallest molecular weight subunit (subunit IV), which contains no redox prosthetic group, is the only supernumerary subunit in the four-subunit Rhodobacter sphaeroides bc1 complex. This subunit is involved in Q binding and the structural integrity of the complex. When the cytochrome bc1 complex is photoaffinity labeled with [3H]azido-Q derivative, radioactivity is found in subunits IV and I (cytochrome b), indicating that these two subunits are responsible for Q binding in the complex. When the subunit IV gene (fbcQ) is deleted from the R. sphaeroides chromosome, the resulting strain (RSdeltaIV) requires a period of adaptation before the start of photosynthetic growth. The cytochrome bc1 complex in adapted RSdeltaIV chromatophores is labile to detergent treatment (60-75% inactivation), and shows a four-fold increase in the Km for Q2H2. The first two changes indicate a structural role of subunit IV; the third change supports its Q-binding function. Tryptophan-79 is important for structural and Q-binding functions of subunit IV. Subunit IV is overexpressed in Escherichia coli as a GST fusion protein using the constructed expression vector, pGEX/IV. Purified recombinant subunit IV is functionally active as it can restore the bc1 complex activity from the three-subunit core complex to the same level as that of wild-type or complement complex. Three regions in the subunit IV sequence, residues 86-109, 77-85, and 41-55, are essential for interaction with the core complex because deleting one of these regions yields a subunit completely or partially unable to restore cytochrome bc1 from the core complex.     

Inhibitory effects of dicyclohexylcarbodiimide on spinach cytochrome b6-f complex

The electron transfer activity of purified cytochrome b6-f complex of spinach chloroplast is inhibited by dicyclohexylcarbodiimide (DCCD) in a concentration and incubation time dependent manner. The maximum inhibition of 75% is observed when 300 mole of DCCD per mole of protein (based on cytochrome f) is incubated with cytochrome b6-f complex at room temperature for 40 min. The inhibition of the complex is not due to the formation of cross links between subunits but due to the modification of carboxyls. The amount of DCCD incorporation is directly proportional to the activity loss, suggesting that some carboxyl groups in the complex are directly or indirectly involved in the catalytic function. The incorporated DCCD is located mainly at cytochrome b6 protein. The partially inhibited complex shows the same H+/e-ratio as that of the intact complex when embedded in phospholipid vesicles.     

Reconstitution of isolated Rieske Fe-S protein into a Rieske-depleted cytochrome b6-f complex

The Rieske Fe-S protein can be isolated from the cytochrome b6-f complex by means of chromatography on a hydroxyapatite column in the presence of detergent. Depletion of the cytochrome complex from the Rieske protein results in the loss of oxidoreductase activity, as well as the ability to reduce cytochrome b6. The Rieske Fe-S protein can be reconstituted into the Rieske-depleted complex by removal of the Triton X-100 molecules associated with the protein fractions, and their substitution by lipids. Upon reconstitution the complex is reactivated, and the role of the Rieske Fe-S protein in the reduction of both plastocyanin and cytochrome b6 can be demonstrated.     

Essentiality of the molecular weight 15,000 protein (subunit IV) in the cytochrome b-c1 complex of Rhodobacter sphaeroides.

The cytochrome b-c1 complex from Rhodobacter sphaeroides was resolved into four protein subunits by a phenyl-Sepharose CL-4B column eluted with different detergents. Individual subunits were purified to homogeneity. Antibodies against subunit IV (Mr = 15,000) were raised and purified. These antibodies had a high titer with isolated subunit IV and with the b-c1 complex from R. sphaeroides. They inhibited 95% of the ubiquinol-cytochrome c reductase activity of the cytochrome b-c1 complex, indicating that subunit IV is essential for the catalytic function of this complex. When detergent-solubilized chromatopores were passed through an anti-subunit IV coupled Affi-Gel 10 column, no no ubiquinol-cytochrome c reductase activity was detected in the effluent, and four proteins, corresponding to the four subunits in the isolated complex, were adsorbed to the column. This indicated that subunit IV in an integral part of the cytochrome b-c1 complex. No change in the apparent Kms for Q2H2 and for cytochrome c was observed with anti-subunit IV treated complex. Antibodies against subunit IV had little effect on the stability of the ubisemiquinone radical in this complex, suggesting that they do not bind to the subunit near its ubiquinone-binding site.     

Rabbit muscle phosphofructokinase. 2. Inactivation by the affinity label 5'-[p-(fluorosulfonyl)benzoyl]-1,N6-ethenoadenosine

The reaction of the fluorescent affinity label 5'-[p-(fluorosulfonyl)benzoyl]-1,N6-ethenoadenosine with rabbit skeletal muscle phosphofructokinase results in an inactivation of the enzyme and in the covalent incorporation of up to one label/monomer. The substrates, MgATP and fructose 6-phosphate, each protect against inactivation of the enzyme, but neither diminishes the extent of covalent incorporation of the label, indicating that the inactivation is not the result of covalent incorporation of the label. Dithiothreitol reactivates the inactivated enzyme but does not reduce the extent of incorporation of the label. A determination of the number of free sulfhydryl groups on the enzyme as a function of the extent of inactivation by the reagent suggests that the inactivation is associated with the loss of two free sulfhydryl groups per phosphofructokinase monomer. The inactivation reaction appears to involve the reversible formation of an enzyme-reagent complex (Kd = 1.11 mM) prior to the conversion of the complex to inactive enzyme (k1 = 0.98 min-1). In view of the protection afforded by either substrate and the evidence suggesting the formation of an enzyme-reagent complex prior to inactivation, it would appear that the inactivation results from a reagent-mediated formation of a disulfide bond between two cysteinyl residues in close proximity, possibly in or near the catalytic site of the enzyme. The site of covalent attachment of the label appears to be the binding site specific for the activating adenine nucleotides cAMP, AMP, and ADP. The extent of covalent incorporation of the label at this site is diminished in the presence of cAMP, and phosphofructokinase modified at this site by this affinity label is no longer subject to activation by cAMP.