Presence Of Reductant Research Articles

Cholate-solubilized erythrocyte glucose transporters exist as a mixture of homodimers and homotetramers

The molecular size of purified, human erythrocyte glucose transport protein (GLUT1) solubilized in cholic acid was determined by size-exclusion chromatography (SEC) and sucrose gradient ultracentrifugation. GLUT1 purified in the presence of dithiothreitol (GLUT1 + DTT) is resolved as a complex of average Stokes' radius 5.74 nm by SEC. This complex displays D-glucose-inhibitable cytochalasin B binding and, upon reconstitution into proteoliposomes, catalyzes cytochalasin B inhibitable D-glucose transport. GLUT1 purified in the absence of dithiothreitol (GLUT1-DTT) is resolved by SEC as at least two particles of average Stokes' radii 5.74 (minor component) and 7.48 nm (major component). Solubilization of GLUT1-DTT in the presence of dithiothreitol reduces the amount of 7.48-nm complex and increases the amount of 5.74-nm complex resolved by SEC. GLUT1-DTT displays D-glucose-inhibitable cytochalasin B binding and, upon reconstitution into proteoliposomes, catalyzes cytochalasin B inhibitable D-glucose transport. Sucrose gradient ultracentrifugation of GLUT1 + DTT in cholate resolves GLUT1 into two components of 4.8 and 7.6 S. The 4.8S complex is the major component of GLUT1 + DTT. The reverse profile is observed upon sucrose gradient ultracentrifugation of GLUT1-DTT. SEC of human erythrocyte membrane proteins resolves GLUT1 as a major broad peak of average Stokes' radius 7.48 nm and a minor component of 5.74 nm. Both components are characterized by D-glucose-inhibitable cytochalasin B binding. Purified GLUT1 is associated with approximately 26 tightly bound lipid molecules per monomer of transport protein. These data suggest that purified GLUT1 exists as a mixture of homodimers and homotetramers in cholate-lipid micelles and that the presence of reductant during solubilization favors dimer formation.

Improvement in the specificity of assays for detection of antibody to hepatitis B core antigen

Reducing agents dramatically alter the specificity of competitive assays for antibody to hepatitis B core antigen (anti-HBc). A specificity improvement was demonstrated with a new assay which utilizes microparticle membrane capture and chemiluminescence detection as well as a current radioimmunoassay procedure (Corab: Abbott Laboratories, Abbott Park, Ill.). The effect was most noticeable with elevated negative and weakly reactive samples. In both systems, reductants increased separation of a negative population (n = 160) from assay cutoffs. With a selected population (n = 307), inclusion of reductant eliminated apparent anti-HBc activity in 54 of 81 samples in the 30 to 70% inhibition range. Reductant-stable anti-HBc samples were strongly associated with the presence of antibody to hepatitis B surface antigen (21 of 27). The association persisted below the detection limits of current assays to 0.3 to 0.4 Paul Ehrlich Institute units per ml. Only 1 of 54 reduction-sensitive borderline samples was confirmed to be positive for antibody to hepatitis B surface antigen. The modified procedures had unchanged or slightly improved sensitivity for immunoglobulin G (IgG)-associated anti-HBc activity. Although IgM anti-HBc detection was reduced from four- to eightfold in the presence of reductants, sensitivities remained at least twofold greater than tha of an enzyme immunoassay (Corzyme M; Abbott) designed to detect acute-phase levels of IgM anti-HBc. The use of reducing agents should significantly improve the reliability of anti-HBc testing, especially near assay cutoffs.

The role of MgATP hydrolysis in nitrogenase catalysis.

Kinetic studies on MgATP hydrolysis by nitrogenase of Azotobacter vinelandii were performed in the presence and in the absence of reducing equivalents. By measuring the ATPase activity of dye-oxidized nitrogenase proteins it can be excluded that reductant-independent ATPase activity is the result of futile cycling of electrons. The turnover rates of MoFe protein during reductant-dependent and reductant-independent ATPase activity, when measured with excess Fe protein, have approximately the same value, i.e. 5 s-1 at pH 7.4 and 22 degrees C, assuming the hydrolysis of four molecules of MgATP per turnover of MoFe protein. For Fe protein on the other hand, the maximum turnover rate during reductant-independent ATPase activity is only about 6% of that of reductant-dependent ATPase activity. While the reductant-dependent ATPase activity shows a sigmoidal dependence on the concentration of MgATP, the reductant-independent ATPase activity yields hyperbolic saturation curves. To account for these results it is proposed that the rate-limiting step during MgATP hydrolysis by oxidized nitrogenase is the rate of regeneration of active Fe protein. In the presence of reductant, the regeneration of active Fe protein is stimulated, explaining the higher ATPase activity of nitrogenase during substrate reduction.

Growth hormone receptor structure in adipocytes from normal and growth hormone-deficient rats.

Using cross-linking techniques, we compared the properties of the growth hormone (GH) receptor in freshly isolated adipocytes from normal rats, from GH deficient rats, and in preincubated adipocytes from normal rats. Bound [125I]iodo-hGH was cross-linked to adipocytes with disuccinimidyl suberate, and membrane proteins labelled with [125I]iodo-hGH were visualized using sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiography. All of the adipocytes tested exhibited a prominent Mr = 134,000 band and additional less intense bands in the presence of reductant. No significant differences in the overall banding pattern of membrane proteins were evident in reducing or nonreducing gels, using adipocytes from rats made GH deficient by hypophysectomy or by treatment with antibodies against rat GH, or in fresh and preincubated cells from normal rats. Taken together with binding studies, these findings suggest that differences in the ability of GH to stimulate glucose oxidation in rat adipose tissue probably involve differences distal to the GH receptor.

Disulfide bonds within the α-subunit of insulin receptors in rat liver and brain membranes

We have characterized inter- and intrasubunit disulfide bonds of insulin receptors using reductant-treated rat liver and brain membranes. In autoradiograms of 125I-insulin cross-linked to both membranes pretreated with dithiothreitol, the intensity of affinity-labeled bands of the αβ-heterodimer and α-subunit was increased. Interestingly, labeled 120 and 110 kDa bands considered to be the α-subunit in partially reduced liver and brain membranes moved to 130 and 120 kDa bands under further reduced conditions, respectively. Double electrophoresis of each partially reduced band in the presence of reductants clearly demonstrates that the α-subunit of insulin receptors contains intrasubunit disulfide bonds.

Electrothermal atomic-absorption and atomic-fluorescence spectrometry with a tungsten-coil atomizer

A pulsed electrothermal atomizer of the tungsten-coil type and apparatus for its application in atomic-absorption and atomic-fluorescence spectrometry are described. A tungsten-coil atomizer is shown to be just as good as commercial electrothermal atomizers with regard to sensitivity and reproducibility, but to have better operating characteristics. A theoretical model for formation of the atom cloud is given. Mechanisms for atomization of different groups of element in an atmosphere of pure argon and in the presence of reductants (hydrogen and carbon) are proposed.

Optimization of hydroxylation of tyrosine and tyrosine-containing peptides by mushroom tyrosinase

Free tyrosine and tyrosine residues in various peptides and proteins are converted into dopa and dopa residues by tyrosinase (monophehol, L-dopa: oxygen oxidoreductase, EC 1.14.18.1) in the presence of reductants. The efficiency of the tyrosine-to-dopa conversion was examined under varied conditions, such as the substrate-to-tyrosine ratio, concentrations of reductant and oxygen in the reaction solution, pH, temperature and reaction time. The highest dopa yields were achieved with the following optimal conditions for hydroxylation: 0.1 M phosphate buffer at pH 7, 25 mM ascorbic acid, 1 mM tyrosine, 50 μg/ml tyrosinase and 20°C. Using these conditions, up to 70% of free tyrosine was converted into dopa, and tyrosine residues in several synthetic peptides were also hydroxylated to dopa residues at ratios as high as free tyrosine. The preparation of hydroxylated analogues of the decapeptide (Ala-Lys-Pro-Ser-Tyr-Pro-Pro-Thr-Tyr-Lys), in particular, may contribute to a better understanding of adhesion in the dopa-containing mussel glue protein

Oxidation of Candutm Fuel by the Products of the Alpha Radiolysis of Groundwater

AbstractThe oxidation of CANDUTM fuel by the products of alpha radiolysis of water is being investigated using X-ray Photoelectron Spectroscopy and electrochemical techniques. Possible mechanisms of oxidation of U02by 02 and H202, two of the oxidants formed during radiolysis of water, are discussed. Oxidation by H202depends upon its concentration and the pH of the solution. The oxidation of U02by the products of alpha radiolysis of water, formed by an alpha flux comparable with that expected at the used- fuel surface at the estimated time of waste container failure, caused very little dissolution of UO2fuel. The presence of reductants in groundwater reaching the fuel surface will diminish the fuel oxidation by the radiolysis products.

Inactivation of red cell glutathione peroxidase by divicine and its relation to the hemolysis of favism

A significant inactivation of red blood cell glutathione peroxidase (25% less than the physiological value) was observed after exposure of intact erythrocytes to 2 mM divicine (an autoxidizable aminophenol from Vicia faba seeds) and 2 mM ascorbate for 3 h at 37°C. Addition of catalase and conversion of Hb to the carbomonoxy derivative resulted in protection against enzyme inactivation. Oxidation of Hb was a concurrent phenomenon, and augmented the inactivating effect. In hemolysates, much stronger effects were observed at shorter times (2 h); divicine was effective also without ascorbate, and the presence of reductants (ascorbate or glutathione or NADPH) enhanced its inactivating power. Of the other antioxidant enzymes, superoxide dismutase was unaffected under the same experimental conditions. Catalase was found to be much less sensitive to the inactivation; it was almost unaffected in experiments with intact erythrocytes and specifically protected by NADPH in experiments with hemolysates. This specific damage of glutathione peroxidase, apparently involving interaction of H 2O 2 and HbO 2, may be related to the pathogenesis of hemolysis in favism.

The nature and regulation of the insulin receptor: structure and function.

Native, cell-surface insulin receptor consists of two glycoprotein subunit types with apparent masses of about 125,000 daltons (alpha subunit) and 90,000 daltons (beta subunit). The alpha and beta subunits are derived from a single polypeptide precursor by one or more proteolytic cleavages. The predominant subunit configuration in the native insulin receptor is a disulfide-linked heterotetrameric structure containing two alpha and two beta subunits. The alpha and beta insulin-receptor subunits seem to have distinct functions such that alpha appears to bind hormone whereas beta appears to possess intrinsic tyrosine kinase activity. In detergent extracts, insulin activates receptor autophosphorylation of tyrosine residues on its beta subunit, whereas in the presence of reductant, the alpha subunit is also phosphorylated. Other physiologically relevant substrates of the insulin receptor tyrosine kinase in target cells, if any, have not yet been identified. In intact cells, insulin activates serine/threonine phosphorylation of insulin receptor beta subunit as well as tyrosine phosphorylation. The biological role of the receptor-associated tyrosine kinase is not known. Tyrosine phosphorylation, catalyzed by either autophosphorylation or purified src kinase, of insulin receptor beta subunit in vitro activates the receptor kinase activity, whereas dephosphorylation with alkaline phosphatase deactivates the receptor kinase. The insulin receptor kinase is regulated by beta-adrenergic agonists and other agents that elevate cAMP in adipocytes, presumably via the cAMP-dependent protein kinase. Such agents decrease receptor affinity for insulin and partially uncouple receptor tyrosine kinase activity from activation by insulin. These effects appear to contribute to the biological antagonism between insulin and beta-agonists. The insulin receptor kinase is also inhibited in intact cells by phorbol esters that mediate serine/threonine phosphorylation of the insulin receptor, presumably via the Ca++-phospholipid-dependent protein kinase. These data suggest the hypothesis that a complex network of tyrosine and serine/threonine phosphorylations on the insulin receptor modulate its binding and kinase activities in an antagonistic manner.

Subunit structure of a high-affinity receptor for type beta-transforming growth factor. Evidence for a disulfide-linked glycosylated receptor complex.

The structure of high-affinity receptors for type beta-transforming growth factor (beta TGF) has been examined by affinity labeling with 125I-beta TGF and disuccinimidyl suberate. The major receptor component labeled by 125I-beta TGF in mouse, rat, and chick fibroblasts migrated as a 280-290-kilodalton species on dodecyl sulfate-polyacrylamide electrophoresis gels in the presence of reductant, dithiothreitol. A larger (330-kilodalton) species was labeled in human fibroblasts, but comparative peptide mapping indicated a close structural relationship with receptors from mouse fibroblasts. In the absence of reductant, the affinity-labeled beta TGF receptor migrated in the gels as a larger disulfide-linked complex. The molecular mass calculated from the hydrodynamic properties of native nonreduced beta TGF receptors was 565 (mouse) or 615 kilodaltons (human). Other molecular parameters for the beta TGF receptor were: Stokes radius, 8.3-8.5 nm; sedimentation coefficient, 12.7-13.0 S; and frictional ratio, f/f0 = 1.4. The beta TGF receptor was solubilized under conditions in which the structural and ligand-binding properties of the native state were retained. beta TGF receptors solubilized from human, mouse, and chick cells interacted specifically with immobilized wheat germ agglutinin. These data suggest that the high affinity receptor for beta TGF in human, rodent, and avian fibroblasts is a disulfide-linked glycosylated 565-615-kilodalton complex with a 280-330-kilodalton subunit that contains the ligand-binding site. The oligomeric structure of the beta TGF receptor does not appear to be induced by receptor occupancy with the ligand.

Ontogeny and characterization of basic somatomedin receptors in rat placenta.

We investigated the binding of [125I]basic somatomedin (B-SM) (insulin-like growth factor-I/SM-C in rat placenta. Low but specific [125I]B-SM binding to placenta was detected in rats from the end of the second week (14 days) of gestation. The binding increased until about 17 days of gestation and then reached a plateau. Binding characteristics did not alter during gestation. In contrast to previous observations [125I]B-SM binding to the tissue was competed more effectively by B-SM than multiplication stimulating activity (a rat counterpart of IGF-II). Insulin competed for the binding at a higher concentration. The Scatchard plot derived from the binding competition data was curvilinear. Autoradiograms of affinity labeled B-SM receptors in placentas at different gestational age subsequent to sodium dodecyl sulfate-gel (7.5%) electrophoresis revealed labeled constituents with approximate mol wt greater than 330 K and 140 K in the presence of reductant and greater than 330 K in the absence of reductant. Detailed analysis of the receptor in term placenta subsequent to 5-13% gradient gel containing sodium dodecyl sulfate revealed two more labeled components (mol wt, 250 K and 36 K) irrespective of the reductant. However, the intensity of the 250 K and 36K was low compared to 140 K and 330 K components. These results suggest that B-SM receptor in rat placenta, similar to human placenta, binds preferentially to type I or 140 K subunit of the 330 K oligomeric SM receptor.

The common α subunit of bovine glycoprotein hormones: Limited formation of native structure by the totally nonglycosylated polypeptide chain

The folding of the bovine glycoprotein hormone alpha subunit, synthesized in bacteria following insertion of the nucleotide sequence coding for this polypeptide, has been studied to determine the effect that a complete lack of carbohydrate has on this process. The bacterially derived alpha polypeptide (bac-alpha), extracted from E. coli in the presence of reductant and denaturant, had an estimated 0.2% native structure as determined by a conformationally sensitive radioimmunoassay. Upon reduction of disulfide bonds and reoxidation in air, the amount of native structure increased about 18-fold. Approximately 2% of the refolded bac-alpha preparation combines with the beta subunit of human chorionic gonadotropin (hCG beta) to form a complex that binds to the gonadotropin receptor and elicits a biological response. Since the correct folding (by immunological criteria) of bac-alpha (ca 3%) is significantly greater than expected from a random formation of disulfide bonds (0.1%), it appears that correct folding of alpha subunit can occur in the complete absence of carbohydrate, though in very low yield. Native bovine lutropin alpha subunit (LH alpha) and chemically deglycosylated LH alpha (which retains two asparagine-linked N-acetyl glucosamine residues per alpha oligosaccharide) were subjected to the same reduction/reoxidation regimen as the bacterially produced alpha subunit. As has been reported previously [Giudice LC, Pierce, JG, J Biol Chem 251: 6392, 1976] intact LH alpha fully regained its native structure. The partially deglycosylated LH alpha also refolds to a native-like structure in high yield as assessed by immunological assays and by its ability to combine with HCG beta to form a biologically active complex. The data show that carbohydrate, while not obligatory for correct folding, greatly facilitates the formation of functional alpha subunit.

Both TSH and thyroid-stimulating antibody of Graves' disease bind to an Mr 197,000 holoreceptor.

Human and porcine thyroid plasma membranes were submitted to sodium-dodecyl sulphate polyacrylamide gel electrophoresis in the absence and presence of reductant and electrotransferred onto nitrocellulose paper. Native bovine TSH, IgG from all 10 patients with active Graves's disease tested, as well as rabbit anti-bTSH anti-idiotypic antibody were found to bind to a single Mr approximately 197,000 band present only in non-reduced gels. We conclude that TSH and Graves' IgG bind to the TSH holoreceptor.

Redox-dependent accessibility of subunit V of cytochrome oxidase. A novel use of ELISA as a probe of intact membranes.

Beef heart cytochrome c oxidase subunit V was isolated by a new one-step procedure. The product was homogeneous to 99% and immunogenically competent. The resulting antibodies inhibited the whole enzyme. A variation of ELISA (enzyme-linked immunosorbent assay) is described which, using a homogeneous antigen, probes the surface of intact membranes to a high degree of specificity. We showed that the subunit is accessible on the surface of inside-out mitochondrial inner membrane particles only when the particles were reduced with ascorbate and N,N,N',N'-tetramethyl-p-phenylene-diamine but is inaccessible on the surface of right-side-out particles regardless of the presence of reductants. These findings have implications with respect to the topography of the enzyme per se and in relation to its neighbors, and with respect to the degree of coupling between electron transport and proton pumping by the enzyme.

Surface labeling of thyrocytes isolated by a new method combining enzymatic digestion and Percoll gradient centrifugation

Thyrocytes were isolated from porcine thyroid glands using a new method entailing two-step collagenase digestion and Percoll gradient centrifugation. Good yield and a high percentage of viabile thyrocytes free from other contaminating cells was achieved. Proteins present on the surface of thyroid cell plasma membranes were then specifically labeled using Iodogen and 125I −. Membrane lysates were separated by electrophoresis on 10% polyacrylamide gel, under reducing and nonreducing conditions, followed by autoradiography. When the gels were stained with silver nitrate some 30 bands were visualized in both the presence and absence of reductant. Only 9 bands were found to be labeled under nonreducing conditions and 12 in the presence of reductant. Two bands involved in the thyrotropin receptor structure— M r = 66,000 and M r = 70,000, respectively—were visualized in the absence of reductant. Upon reduction the M r 66,000 band was retained and a new band ( M r = 33,000) was seen. The mild enzymatic treatment used in isolating thyrocytes and the lack of contamination with other cells allowed the consistent labeling of exposed plasma membrane components by the Iodogen method such that the orientation of thyrotropin receptor components in the plasma membrane could be deduced.

Nickel containing hydrogenases

Hydrogenases have been purified from different biological sources. They are highly diversified enzymes in terms of active centers constitution, although they catalyze the simplest oxidation-reduction process: H 2 ⇌ 2H + + 2e. Hydrogenases have been recognized so far to be ironsulfur proteins. Generally they contain from four to twelve atoms of non-haem iron arranged in FeS clusters representative of the known basic structures, e.g., [2Fe-2S], 3Fe-xS], and [4Fe-4S] [1–7]. Recently, nickel joined the group of transition metals relevant in biological oxidation-reduction ▪ processes. It was shown to be a structural component of the hydrogenases isolated from Desulfovibrio gigas [1, 2], Desulfovibrio desulfuricans (ATTC 27774) [3], Desulfovibrio desulfuricans (Norway strain) [4], Methanosarcina barkeri [5], Methanobacterium thermoautotrophicum [6] and Chromatium vinosum [8]. With the exception of the last one, they were demonstrated to contain EPR nickel redox dependent signals. As an example, D. gigas hydrogenase exhibits rhombic EPR signals, with g-values 2.31, 2.23 and 2.02 (see Fig. 1). Using isotopic reconstitution by 61Ni (nuclear magnetic moment I = 3 2 ), the EPR signal was proven arise from a nickel species [9]. The same types of experiments were reported for M. thermoautotrophicum [6] and D. desulfuricans (ATCC 27774) hydrogenases [3]. A detailed EPR study on the oxidation-reduction transition of the EPR detectable species in the presence of reductant (dithionite and hydrogen) indicates [1, 2]: 1. The reduction of the Ni EPR active species is an one-electron process (possibly associated with the redox couple Ni(III)Ni(II)). 2. No evidence was found so far for exchangeable protons in the vicinity of the nickel center in the oxidized (native) state. However, hydrogen reduced samples originate a different EPR rhombic Ni signal, which may represent an active transient species occurring during the activation of hydrogen molecules [9]. Thus, it is attractive to propose the presence of a hydride intermediate in analogy with nickel catalysts involved in hydrogenation processes [10]. 3. Although the determined mid-point redox potential (−220 mV) is more negative than that expected for nickel compounds [11] it is still more positive than that of the substrate couple H 2/H +. The value determined was shown to be pH dependent [2].

The isolation of alpha-1-protease inhibitor by a unique procedure designed for industrial application

Individuals who are congenitally deficient in the human plasma protein α 1-protease inhibitor ( α 1PI, which is also called α 1-antitrypsin) usually develop chronic obstructive lung disease as a consequence of improperly regulated granulocyte elastase. In this report, a unique, facile one- or two-step method is presented for the large-scale isolation of α 1PI for potential therapeutic use. The method takes advantage of the unusual disulfide bond in α 1PI, which consists of a single cysteine residue in the polypeptide chain bound to a free pendant cysteine. In contrast to other circulating plasma proteins, the disulfide bridge in α 1PI does not add to its structural stability. Therefore, if an α 1PI-containing solution of plasma proteins is precipitated out in the presence of reductant, much more extensive separation of contaminating proteins will be achieved than in the absence of such reductant. We have used Cohn Fraction IV-1, a relatively unused side product in albumin and gamma globulin production, as our starting material. After activation of the α 1PI in basic media, partial purification is achieved with successive additions of Aerosil (a fumed silica), dithiothreitol, and ammonium sulfate. From 90 to 95% of the contaminating proteins are precipitated by this single procedure, resulting in a product that is ∼70% pure. DEAE-cellulose chromatography can be used as an additional purification step, and this results in a product that is nearly homogenous. Overall yield is ∼45%. The method is simple, inexpensive, and reproducible and is directly applicable to large-scale industrial processing.

H 2O 2− and alkyl hydroperoxide-supported para hydroxylation of aniline by alkaline hematin

Under alkaline conditions and in the presence of reductant, NADH or NADPH, and molecular oxygen, hemin catalyzes the regiospecific para-hydroxylation of aniline to form p-aminophenol [P.A. Adams, D.A. Baldwin, and M.C. Berman, J. Chem. Soc. ( Lond) Chem. Commun. 856 (1979); P.A. Adams and M.C. Berman, J. Inorg. Biochem. 17, 1 (1982)]. The reaction has now been studied in the presence of H 2O 2 and alkyl hydroperoxides and in the absence of oxygen and reductant. Results indicate that the H 2O 2 − and alkyl hydroperoxide-supported processes proceed via different mechanisms involving, on the one hand, the hydroperoxide anion (HO 2 −) and on the other, the undissociated alkyl hydroperoxide molecule (ROOH). The addition of superoxide dismutase to the reaction had no effect, unlike the NADH/O 2 supported reaction where the enzyme completely inhibits reaction. Similarities between the hemin-mediated peroxide-supported reactions reported here, and the cytochrome P-450-mediated peroxide-supported reactions reinforce our earlier contentions that the alkaline hemin system appears to be a good model for the in vivo activation of oxygen by hemoproteins.

On the mechanism of the cytochrome c oxidase reaction. Bound Fe(III) cytochrome c as an intermediate in the reductive half reaction.

The reaction between cytochrome c and Cytochrome aa3 in the presence of reductant was studied using transient and steady‐state kinetic methods. To investigate the effect of complex formation the reaction was studied at both high and low ionic strength in potassium phosphate buffer (pH 7.4). Steady‐state kinetic experiments were performed in which the oxidation level of cytochrome c was measured during the anaerobic‐aerobic transition of a system containing cytochrome aa3, cytochrome c and ascorbate. Such measurements confirm previous suggestions [Mochan and Nicholls (1972) Biochim, Biophys. Acta. 172, 37–51] that cytochrome c forms a complex with cytochrome oxidase which is not easily accessible to ascorbate reduction. The results suggest that one oxidised cytochrome c per oxidase is bound at low ionic strength. The reduction of ‘oxygen‐pulsed’ oxidase in the presence of dithionite and various concentrations of cytochrome c was investigated by stopped flow spectrophotometry. Exposure of buffer containing cytochrome aa3, cytochrome c and dithionite to a brief pulse of oxygen caused an immediate oxidation of the oxidase and of up to one equivalent of cytochrome c per oxidase. Under conditions where complex formation is favoured the subsequent reduction of cytochrome c is many times slower than can be accounted for by the reaction between dithionite and free cytochrome c. This suggests that the bound oxidised cytochrome c is not readily accessible to dithionite reduction. It is proposed that the complex between oxidised cytochrome c and the oxidase is an intermediate of the reductive half‐reaction and it is shown that the kinetic responses of the complex are rapid enough to be in agreement with this suggestion.