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The hypertensive effect observed with most cell-free haemoglobins has been proposed to result from NO scavenging. However, a newly developed PEG [poly(ethylene glycol)]-conjugated haemoglobin, MalPEG-Hb [maleimide-activated PEG-conjugated haemoglobin], is non-hypertensive with unique physicochemical properties: high O2 affinity, low co-operativity and large molecular radius. It is therefore of interest to compare the ligand-binding properties of MalPEG-Hb with unmodified cell-free HbA (stroma-free human haemoglobin). NO association rates for deoxy and oxyMalPEG-Hb and HbA were found to be identical. These results confirm the lack of correlation between hypertension and NO for a similar modified haemoglobin with high molecular radius and low p50 (pO2 at which haemoglobin is half-saturated with O2) [Rohlfs, Bruner, Chiu, Gonzales, Gonzales, Magde, Magde, Vandegriff and Winslow (1998) J. Biol. Chem. 273, 12128–12134]. The R-state O2 association kinetic constants were also the same for the two haemoglobins. However, even though the p50 of MalPEG-Hb is approx. half of that of HbA, the biphasic O2 dissociation rates measured at relatively high pO2 (150 Torr) were 2-fold higher, giving rise to a 2-fold lower R-state equilibrium association constant for MalPEG-Hb compared with HbA. Thus the O2 affinity of MalPEG-Hb is higher only at pO2 values lower than the intersection point of the O2 equilibrium curves for MalPEG-Hb and HbA. In summary, the present studies found similar rates of NO binding to HbA and MalPEG-Hb, eliminating the possibility that the lack of vasoactivity of MalPEG-Hb is simply the result of reduced molecular reactivity with NO. Alternatively, the unique O2-binding characteristics with low p50 and co-operativity suggest that the ‘R-state’ conformation of MalPEG-Hb is in a more T-state configuration and restricted from conformational change.

Here we report the structural and functional characterization of a covalent complex (MKP) obtained by cross-linking microperoxidase (Mp), the haem-undecapeptide obtained by the peptic digestion of cytochrome c, with a 21-residue synthetic peptide (P21) analogous to the S-peptide of the RNase A. The covalent complex has been prepared by introducing a disulphide bond between Cys-1 of P21 and Lys-13 of Mp, previously modified with a thiol-containing reagent. On formation of the complex (which is a monomer), the helical content of P21 increases significantly. The results obtained indicate that His-13 of P21 co-ordinates to the sixth co-ordination position of the haem iron, thus leading to the formation of a complex characterized by an equilibrium between an ‘open’ and a ‘closed’ structure, as confirmed by molecular dynamics simulations. Under acidic pH conditions, where His-13 of P21 is loosely bound to the haem iron (‘open’ conformation), MKP displays appreciable, quasi-reversible electrochemical activity; in contrast, at neutral pH (‘closed’ conformation) electrochemical behaviour is negligible, indicating that P21 interferes with the electron-transfer properties typical of Mp. On the whole, MKP is a suitable starting material for building a miniature haem system, with interesting potential for application to biosensor technology.

Thermodynamic and kinetic properties of O2 and CO binding to haemoglobin (Hb) Kempsey [Asp-G1(99) beta----Asn] were investigated and the activation parameters for the two ligands were determined. At every temperature the O2-binding isotherms display a weak co-operativity, n ranging between 1.1 and 1.2, and dissociation kinetics show a single-exponential behaviour. O2-binding kinetics were studied at 25 degrees C by temperature jump and are characterized at each saturation (from Y = 0.31 to Y = 1.0) by two processes, a fast bimolecular one and a slow monomolecular one (tau -1 = 20 s-1), which contributes to approx. 30% of the whole relaxation amplitude at every Y. CO-binding kinetics to Hb Kempsey were followed at several temperatures by flash photolysis and stopped flow. The process is biphasic, as reported elsewhere [Bunn, Wohl, Bradley, Cooley & Gibson (1974) J. Biol. Chem. 249, 7402-7409], and the relative contributions of the two bimolecular rates to the whole process are only slightly affected by temperature. On taking account for the fraction of dimers at every protein concentration, the slow phase corresponds to approx. 50% of the ligand binding to tetramers. Correlation of these results with previous spectroscopic data leads to the hypothesis that the biphasic time course of CO binding may be attributed to alpha/beta heterogeneity of the R-state of tetrameric Hb Kempsey.

The c.d. spectrum of oxyhaemoglobin from Camelus dromedarius is significantly affected by the presence of inositol hexakisphosphate. Correlation with O2-binding measurements shows that these dichroic changes parallel the functional properties of the protein. The optical modifications suggest that, in contrast with human haemoglobin, the conformational changes induced by inositol hexakisphosphate on dromedary oxyhaemoglobin are mainly attributable to a local change of the tertiary structure reminiscent of that of the deoxy derivative, the quaternary conformation seeming to be almost unaffected. The results provide direct evidence of the existence on the protein of two distinct sites for polyanions.

The kinetics of oxidation of azurin and cytochrome c-551 catalysed by Pseudomonas aeruginosa cytochrome oxidase were re-investigated, and the steady-state parameters were evaluated by parametric and non-parametric methods. At low concentrations of substrates (e.g. less than or equal to 50 microM) the values obtained for Km and catalytic-centre activity are respectively 15 +/- 3 microM and 77 +/- 6 min-1 for azurin and 2.15 +/- 0.23 microM and 66 +/- 2 min-1 for cytochrome c-551, in general accord with previous reports assigning to cytochrome c-551 the higher affinity for the enzyme and to azurin a slightly higher catalytic rate. However, when the cytochrome c-551 concentration was extended well beyond the value of Km, the initial velocity increased, and eventually almost doubled at a substrate concentration greater than or equal to 100 microM. This result suggests a ‘half-hearted’ behaviour, since at relatively low cytochrome c-551 concentrations only one of the two identical binding sites of the dimeric enzyme seems to be catalytically active, possibly because of unfavourable interactions influencing the stability of the Michaelis-Menten complex at the second site. When reduced azurin and cytochrome c-551 are simultaneously exposed to Ps. aeruginosa cytochrome oxidase, the observed steady-state oxidation kinetics are complex, as expected in view of the rapid electron transfer between cytochrome c-551 and azurin in the free state. In spite of this complexity, it seems likely that a mechanism involving a simple competition between the two substrates for the same active site on the enzyme is operative. Addition of a chemically modified and redox inactive form of azurin (Hg-azurin) had no effect on the initial rate of oxidation of either azurin and cytochrome c-551, but clearly altered the time course of the overall process by removing, at least partially, the product inhibition. The results lead to the following conclusions: (i) reduced azurin and cytochrome c-551 bind at the same site on the enzyme, and thus compete; (ii) Hg-azurin binds at a regulatory site, competing with the product rather than the substrate; (iii) the two binding sites on the dimeric enzyme, though intrinsically equivalent, display unfavourable interactions. Since water is the product of the reduction of oxygen, point (iii) has important implications for the reaction mechanism.

Amino acid analyses and peptide mapping were performed for the four main haemoglobins from the armoured catfish Pterygoplichthys pardalis; component I, which is functionally distinct from the others, is structurally unique, whereas components II, III and IV, functionally indistinguishable, are closely related in structure. Compositional difference indices are calculated for the four components and for the two major haemoglobins from the trout Salmo irideus, and the results are discussed in terms of structural relationships and evolutionary history of fish haemoglobins.

The redox reaction between cytochrome c-551 and its oxidase from the respiratory chain of pseudomonas aeruginosa was studied by rapid-mixing techniques at both pH7 and 9.1. The electron transfer in the direction of cytochrome c-551 reduction, starting with the oxidase in the reduced and CO-bound form, is monophasic, and the governing bimolecular rate constants are 1.3(+/- 0.2) x 10(7) M-1 . s-1 at pH 9.1 and 4 (+/- 1) x 10(6) M-1 . s-1 at pH 7.0. In the opposite direction, i.e. mixing the oxidized oxidase with the reduced cytochrome c-551 in the absence of O2, both a lower absorbance change and a more complex kinetic pattern were observed. With oxidized azurin instead of oxidized cytochrome c-551 the oxidation of the c haem in the CO-bound oxidase is also monophasic, and the second-order rate constant is 2 (+/- 0.7) x 10(6) M-1 . s-1 at pH 9.1. The redox potential of the c haem in the oxidase, as obtained from kinetic titrations of the completely oxidized enzyme with reduced azurin as the variable substrate, is 288 mV at pH 7.0 and 255 mV at pH 9.1. This is in contrast with the very high affinity observed in similar titrations performed with both oxidized azurin and oxidized cytochrome c-551 starting from the CO derivative of the reduced oxidase. It is concluded that: (i) azurin and cytochrome c-551 are not equally efficient in vitro as reducing substrates of the oxidase in the respiratory chain of Pseudomonas aeruginosa; (ii) CO ligation to the d1 haem in the oxidase induces a large decrease (at least 80 mV) in the redox potential of the c-haem moiety.

1. The catalytic cycle of the haem-containing nitrite reductase (NIR) from Pseudomonas aeruginosa involves electron transfer between the two prosthetic groups of the enzyme, the c-haem and the d1-haem; this reaction was shown to be slow by stopped-flow analysis. The recombinant enzyme, expressed in Pseudomonas putida, contains the c-haem but no d1-haem; we have reconstituted this protein with Zn-protoporphyrin IX in the place of the d1-haem. 2. Photoexcitation of Zn-NIR is followed by electron transfer from the triplet excited state of the Zn-porphyrin to the oxidized c-haem, with a rate constant of 7×105 s-1; since the intermediate with reduced c-haem is not significantly populated, we conclude that the back reaction is probably as fast. 3. Even taking into account that in the native NIR the driving force is close to zero, the rate constant for the c → d1 electron transfer, estimated from our experiments, is still much higher than that observed by stopped flow (k = 0.3 s-1) using reduced azurin as the electron donor. This finding may be a direct kinetic indication that reduction of the d1-haem is associated with a substantial reorganization of the co-ordination of the metal, as shown by spectroscopy of the oxidized and reduced NIR.

The electron-transfer reactions of cellobiose oxidase (CBO) have been investigated by conventional and by rapid-scan stopped-flow spectroscopy at pH 6.0. Analysis of the absorbance/time/wavelength matrix by Singular Value Decomposition (SVD) confirms earlier studies showing that cellobiose rapidly reduces the flavin group (7.7 s-1; cellobiose, 100 microM) which in turn slowly (0.2 s-1) reduces the cytochrome b moiety. In the presence of CBO, cellobiose reduces cytochromes c in a reaction that does not depend on oxygen or superoxide. The rate limit for this process is independent of the source of the cytochromes c and is identical with the rate of cytochrome b reduction. Rapid-mixing experiments show that cytochrome b may donate electrons very rapidly to either mammalian cytochrome c or bacterial cytochrome c-551. The reactions were second-order (kc = 1.75 x 10(7) M-1 x s-1; kc-551 = 4.3 x 10(6) M-1 x s-1; pH 6.0, 21 degrees C and I0.064) and strongly ionic-strength (I)-dependent: kc decreasing with I and kc-551 increasing with I. These results suggest the electron-transfer site near cytochrome b bears a significant negative charge. Equilibrium gel chromatography confirms that CBO oxidase and positively charged mammalian cytochrome c make stable complexes. These results are discussed in terms of a model suggesting an electron-transfer role for cytochrome b in vivo, possibly connected with radical-mediated cellulose breakdown.

Nitrite reductase from Pseudomonas aeruginosa has been successfully expressed in Pseudomonas putida. The purified recombinant enzyme contains haem c but no haem d1. Nonetheless, like the holoenzyme from Ps. aeruginosa, it is a stable dimer (molecular mass 120 kDa), and electron transfer to oxidized azurin is biphasic and follows bimolecular kinetics (k1 = 1.5 x 10(5) and k2 = 2.2 x 10(4) M-1.s-1). Unlike the chemically produced apoenzyme, recombinant nitrite reductase containing only haem c is water-soluble, stable at neutral pH and can be quantitatively reconstituted with haem d1, yielding a holoenzyme with the same properties as that expressed by Ps. aeruginosa (namely optical and c.d. spectra, molecular mass, cytochrome c551 oxidase activity and CO-binding kinetics).

Cytochrome c oxidase, after reconstitution into phospholipid vesicles, displays respiratory control. This appears as an inhibition of substrate oxidation (cytochrome c) or reduction (O2) rates which, in the first few turnovers, can be largely removed upon addition of valinomycin, a specific K+ carrier. We report experiments designed to measure directly the internal electron transfer leading to the reduction of cytochrome a3/CuB, in the presence and the absence of a membrane potential. The results suggest that, after the complete oxidation and partial re-reduction of the protein, electron transfer to the binuclear site is valinomycin-sensitive, i.e. is inhibited by the membrane potential. The first-order rate constants calculated in the absence and presence of valinomycin were 0.5-0.6 and 5-6 s-1 respectively. Kinetic analysis of the reduction process is consistent with the conclusion that the membrane potential is below the critical threshold until the first electron is transferred to the cytochrome a3/CuB site. Furthermore, the respiratory control ratio obtained from the dependence of the internal electron transfer rate constant on valinomycin is always higher (by factor of 2) than that measured under turnover conditions either polarographically or spectrophotometrically. Two possible interpretations of this discrepancy are discussed.

The reaction of ascorbate-reduced Pseudomonas cytochrome oxidase with oxygen was studied by using stopped-flow techniques at pH 7.0 and 25 degrees C. The observed time courses were complex, the reaction consisting of three phases. Of these, only the fastest process, with a second-order rate constant of 3.3 × 10(4) M-1.S-1, was dependent on oxygen concentration. The two slower processes were first-order reactions with rates of 1.0 +/- 0.4s-1 and 0.1 +/- 0.03s-1. A kinetic titration experiment revealed that the enzyme had a relatively low affinity constant for oxygen, approx. 10(4)M-1. Kinetic difference spectra were determined for all three reaction phases, showing each to have different characteristics. The fast-phase difference spectrum showed that changes occurred at both the haem c and haem d1 components of the enzyme during this process. These changes were consistent with the haem c becoming oxidized, but with the haem d1 assuming a form that did not correspond to the normal oxidized state, a situation that was not restored even after the second kinetic phase, which reflected further changes in the haem d1 component. The results are discussed in terms of a kinetic scheme.

By enzymic digestion of the polysaccharide part of the covalent complex between cytochrome c and Sephadex G-200, a new water-soluble cytochrome c derivative is obtained (called cytochrome cr). Measurement of the free amino groups of this derivative indicates that on average the molar ratio between cytochrome c and polysaccharide is close to 1. Chemical determination of the sugar content gives a value of approx. 24000 for the molecular weight of cytochrome cr. On these bases the soluble cytochrome cr complex may be thought of as a folded protein to which a long polysaccharide tail is covalently bound. The functional behaviour of cytochrome cr is much more similar to that of the native molecule than to that of the insoluble complex (cytochrome ci). In particular the kinetics of the reaction of cytochrome cr and cytochrome cn (native) with ascorbate, ferrocyanide-ferricyanide, O2 and cytochrome c oxidase were investigated in considerable detail. The results of these experiments, together with the observation that the insoluble complex of cytochrome c is a very poor substrate of cytochrome c oxidase [Colosimo, Brunori & Antonini (1976) Biochem. J. 153, (657-661], indicate that hindrance effects constraining the approach between cytochrome cr and its oxidase are of greater importance than specific chemical modifications in determining the functional behavior of the protein.

The electron-transfer reaction between azurin and the cytochrome oxidase from Pseudomonas aeruginosa was investigated by temperature-jump relaxation in the absence of O2 and in the presence of CO. The results show that: (i) reduced azurin exists in two forms in equilibrium, only one of which is capable of exchanging electrons with the Pseudomonas cytochrome oxidase, in agreement with M. T. Wilson, C. Greenwood, M. Brunori & E. Antonini (1975) (Biochem. J. 145, 449-457); (ii) the electron transfer between azurin and Pseudomonas cytochrome oxidase occurs within a molecular complex of the two proteins; this internal transfer becomes rate-limiting at high reagent concentrations.

1. The decay of oxyferroperoxidase to ferriperoxidase was studied by rapidly mixing solutions of ferroperoxidase with various amounts of oxygen and following the time-course of appearance of oxyferroperoxidase and its subsequent decay to ferriperoxidase by reaction with ferroperoxidase. 2. The scheme can be accommodated by and [Formula: see text] and occurs without detectable intermediates being observed in the millisecond time-range. 3. Analogue-computer simulation of the reaction is in agreement with the initial rate-limiting reaction being an intermolecular, not intramolecular, electron-donating process, and analysis of the data leads to quantitative values for the rate constants of the overall process. 4. The reaction of oxyferroperoxidase with ferroperoxidase is a model of a terminal oxidase, and the results are discussed in terms of the possible importance of this reaction in peroxidase function, and also in the light it throws on autoxidation of haem compounds.

Studies on horse heart cytochrome c polymers were carried out by stopped-flow and photolysis techniques, to investigate the properties of the CO complex and the kinetics of electron transfer, mainly of the dimeric and tetrameric forms. CO binding, which does not occur with native monomers, proceeds at both pH7.0 and pH9.6, and appears to follow complex kinetics: an initial phase is observed, which is CO-concentration-dependent, followed by a very slow monomolecular phase (k~2×10-3s-1 at pH7) before establishment of equilibrium. Photodissociation of the CO complex has a very low quantum yield, probably less than 0.1. Static titration data of the dimer gave an ‘n’ value of 0.4. These data strongly suggest heterogeneity of the population of binding sites, and have been interpreted in terms of the existence of different structures, probably owing to the non-unique type of binding of monomers during polymerization. Polymers of cytochrome c carboxymethylated on the methionine residue normally ligated to iron show simple CO recombination kinetics after photolytic removal (kon=1.5×106m-1·s-1 at pH6). We therefore suggest that, for native cytochrome c, polymerization has an effect on the lability of the haem crevice, rendering the iron available for binding ligands, without, however, forming the structure of a truly open crevice. Electron transfer is, on the other hand, a simple process, and no gross differences are observed between monomer and polymers. A simple model, taking into account all these data, is suggested.

A covalent complex between cytochrome c oxidase and Saccharomyces cerevisiae iso-1-cytochrome c (called caa3) has been prepared at low ionic strength. Subunit III Cys-115 of beef heart cytochrome c oxidase cross-links by disulphide bond formation to thionitrobenzoate-modified yeast cytochrome c, a derivative shown to bind into the high-affinity site for substrate [Fuller, Darley-Usmar and Capaldi (1981) Biochemistry 20, 7046–7053]. Stopped-flow experiments show that (1) covalently bound yeast cytochrome c cannot donate electrons to cytochrome oxidase, whereas oxidation of exogenously added cytochrome c and electron transfer to cytochrome a are only slightly affected; (2) the steady-state reduction levels of cytochrome c and cytochrome a in the covalent complex caa3 are higher than those found in the native aa3 enzyme. However, (3) Km and Vmax values obtained from the non-linear Eadie–Hofstee plots are very similar in both caa3 and aa3. The results imply that cytochrome c bound to the high-affinity site is not in a configuration optimal for electron transfer.

The cyanide ion is a ligand of ferrous as well as ferric haemoproteins and this study presents a kinetic characterization of the dissociation of its complexes with the two main haemoglobin components from trout blood. Both these haemoglobins bind oxygen co-operatively at neutral or alkaline pH values but one of them is insensitive to pH and allosteric effectors (haemoglobin I, HbI) while the other (haemoglobin IV, HbIV) is strongly sensitive and shows the so-called Root effect (i.e. the incomplete oxygen saturation in air-equilibrated solutions at pH values of < 6.5). Comparison of the kinetics of dissociation of cyanide from ferrous forms of HbI and HbIV reveals that: (i) cyanide dissociates in both cases by a complex reaction, and, at least in the case of HbIV, this may be attributed to functional differences between the α and β subunits; (ii) the reaction is only scarcely co-operative in HbI and not at all so in HbIV; and (iii) the Bohr and Root effects are not manifested in this reaction. The functional heterogeneity of ferrous α and β chains of trout HbI has not been observed for any other ligand; moreover, the observation that co-operativity for cyanide dissociation is expressed by human haemoglobin but not by trout HbIV is surprising.

The isolation and cloning of the cDNA coding for myoglobin (Mb) from the mollusc Aplysia limacina is reported here. Five amino acid differences from the previously published protein sequence have been found in positions 22, 26, 27, 77 and 80 by back translating the cDNA; some of these may be relevant for overall structure stabilization in this Mb. High-level expression of the holoprotein in Escherichia coli has been achieved in the presence of the haem precursor δ-aminolevulinic acid, underlying the importance of tuning haem and apoprotein biosynthesis to achieve high-level expression of haemproteins in bacteria. The recombinant protein is identical to the protein purified from the mollusc buccal muscle. Native A. limacina Mb has an oxygen dissociation rate constant of 70 s-1 [as compared with the value of 15 s-1 for sperm whale Mb, which displays His(E7) and Thr(E10)] (amino acid positions are referred to within the eight helices A–H of the globin fold). In order to understand the mechanism of oxygen stabilization in A. limacina Mb, we have prepared and investigated three active-site mutants: two single mutants in which Val(E7) and Arg(E10) have been replaced by His and Thr, respectively, and a double mutant carrrying both mutations. When Arg(E10) is substituted with Thr, the oxygen dissociation rate constant is increased from 70 s-1 to more than 700 s-1, in complete agreement with the previously proposed role of the former residue in ligand stabilization. In the His(E7)-containing single and double mutants, both displaying high oxygen dissociation rates, the stabilization of bound oxygen by the distal His is insufficient to slow down the ligand dissociation rate constant to the value of sperm whale Mb. These results essentially prove the hypothesis that in A. limacina Mb a mechanism of oxygen stabilization involving Arg(E10), and thus different from that mediated by His(E7), has evolved.

Complexes of cytochrome c oxidase and cytochrome c (Fe- or Zn-containing) have been prepared by 1-ethyl-3-[3-(dimethylamino)propyl]carbodi-imide (EDC) cross-linking. The site to which the cytochrome c covalently binds has been identified as being the same, or close to, the site occupied by cytochrome c in the electrostatic complex which may be formed between the proteins. Stopped-flow experiments, monitored either at a single wavelength or through a rapid wavelength-scan facility, showed that covalently bound Fe-containing cytochrome c cannot donate electrons to cytochrome a. Free Fe-containing cytochrome c was, however, able to transfer electrons to cytochrome a in covalent complexes containing either Fe- or Zn-containing cytochrome c. Turnover experiments showed that the complexed enzyme remains catalytically competent but with decreased (40-80%) activity. The steady-state levels of reduction of both free cytochrome c and cytochrome a in the covalent complex were higher than found in the control (uncomplexed) enzyme. These results are discussed with reference to the structure of the covalent complex and lead us to conclude that cytochrome a may accept electrons directly from free cytochrome c and that cross-linking impairs the redox properties of the CuA site.

The direct electrochemistry of cytochrome c at a gold electrode was investigated by cyclic voltammetry using, as promoters, microperoxidase (the haem-undecapeptide obtained by hydrolysis of cytochrome c), Fe(III)-protoporphyrin IX or protoporphyrin-IX, all entrapped in a cellulose triacetate membrane. The results indicate that these immobilized systems strongly enhance the rate of electron transfer between the protein in solution and the electrode surface, and thus behave as ‘solid-state’ promoters, though with differing efficiencies. These results are of interest because they raise the possibility of engineering an efficient and versatile promoter active also at inert electrode surfaces.

The effect of gangliosides on membrane permeability was investigated by studying the kinetic properties of cytochrome c oxidase, the activity of which, when the enzyme is reconstituted in phospholipid vesicles, is dependent on membrane permeability to H+ and K+. The experiments indicate that three different gangliosides (GM1, DD1a, GT1b) incorporated into cytochrome c oxidase-containing phospholipid vesicles stimulate enzymic activity, in the absence of ionophores, most probably by disorganizing the bilayer lipid assembly and increasing its permeability to ions. This interpretation was confirmed by fluorescence-spectroscopy experiments in which the rate of passive leakage of carboxyfluorescein entrapped in the vesicles was measured. Cholera toxin, or its isolated B-subunit, added to GM1-containing proteoliposomes inhibited cytochrome c oxidase activity, indicating the lack of formation, under these experimental conditions, of channels freely permeable to H+ or K+.

The spectral (e.p.r. and absorbance) properties of the NO and deoxy derivatives of ferrous horseradish peroxidase (HRP; EC 1.11.1.7) and baker's-yeast cytochrome c peroxidase (CCP; EC 1.11.1.5) were investigated between pH 7 and pH 2; over the same pH range the kinetics for CO binding were also determined. At neutral pH the e.p.r. and absorption spectra of the NO and deoxy derivatives of HRP and CCP are typical of systems in which the haem iron is in the hexaco-ordinated state and the pentaco-ordinated state respectively. By lowering pH, the e.p.r. and absorption spectra of HRP and CCP undergo reversible transitions, with pKa values of 4.1 for the NO derivatives and less than or equal to 3 for the deoxy derivatives of the ferrous forms. By analogy with O2-carrying proteins and haem model compounds, the pH-dependent spectral changes of HRP and CCP were interpreted as indicative of the protonation of the N(epsilon) atom of the proximal histidine residue and of the cleavage of the Fe-N(epsilon) bond. However, the slow second-order rate constant (0.003 microM-1.s-1) for CO binding to deoxy ferrous HRP and CCP does not increase substantially even at pH 2.6, suggesting that changes in the Fe-haem plane geometry, presumably associated with the cleavage of the Fe-N(epsilon) bond, do not affect appreciably the observed ligand association rate constant.

The activity of cytochrome oxidase reconstituted into phospholipid vesicles has been studied as a function of orthophosphate, ATP and inositol hexakisphosphate concentrations. The respiratory-control ratio was found to be quite sensitive to these compounds and was inversely related to the anion concentration. This effect is related to a phosphate-dependent decrease in the rate constant for ferrocytochrome c oxidation observed in the presence of ionophores. The data cannot be interpreted simply on the basis of ionic strength, which is known to limit cytochrome c binding to cytochrome oxidase, since cytochrome oxidase-containing vesicles responded differently to phosphate depending on the energization state of the phospholipid membrane.

The role of chloride ions in modulating polyanion-induced conformational changes in haemoglobin from the dromedary (Camelus dromedarius) has been investigated. The results obtained have shown that: in the ferric derivative at pH 6.5 the effect of single polyanion (dextran sulphate and inositol hexakisphosphate) on the conformation is essentially local, thus involving only the tertiary structure of the protein; the presence of chloride ions at a concentration close to the physiological value (i.e. 150 mM) is essential to induce quaternary conformational changes in the polyanion-ferric protein system; comparison between structural and functional data correlates polyanion-induced tertiary conformational changes with changes in the value of midpoint potential, E'0, and quaternary changes with co-operativity.

The effect of inositol hexakisphosphate on the redox equilibria and on the c.d. spectra of ferric derivatives of haemoglobin from Camelus dromedarius has shown that: two distinct functionally relevant binding sites for polyanions are present on the protein; conformational changes promoted by inositol hexakisphosphate are largely dependent on spin state of the iron; tertiary and quaternary changes are not necessarily linked; structures induced by polyanions can be mixed forms that are neither T-state nor R-state.

Cytochrome c oxidase from ox heart was depleted of subunit III and its transient kinetic properties studied by stopped-flow and flash photolysis. It was found that the overall mechanism of electron transfer is very similar for subunit-III-depleted and native oxidase, although significant differences in some kinetic parameters have been detected. These include the second-order rate constant for cytochrome c oxidation and the rate-limiting step of the overall process. Moreover, at low cytochrome c/oxidase ratios (where the number of reducing equivalents is insufficient), the rate of reoxidation of cytochrome a was found to be very slow, even in air, and in fact for the subunit-III-depleted enzyme is even slower than for the native oxidase. The stability of reduced cytochrome a excludes the likelihood that removal of subunit III leads to a new O2-binding site, and the result may be relevant to the lowered vectorial H+/e- stoichiometry. The subunit-III-depleted oxidase can be pulsed under appropriate conditions and its combination with CO is unchanged, as shown by kinetic experiments and difference spectroscopy.

The reaction between lentil (Lens culinaris) seedling amine oxidase and its chromogenic substrate, p-dimethylaminomethylbenzylamine, has been studied by the stopped-flow technique. Upon being mixed with substrate in the absence of oxygen, the enzyme is bleached in a complex kinetic process. A yellow intermediate absorbing at 464 nm and the first product (aldehyde) are formed in subsequent steps. When oxygenated buffer is mixed with substrate-reduced amine oxidase, the 496 nm absorption of the oxidized enzyme is very rapidly restored in a second-order process (k = 2.5 × 10(7) M-1 × S-1). This reaction is appreciable even at very low oxygen concentration, in keeping with the fairly low Km for O2 measured by steady-state kinetics.

Experiments were performed to examine the cyanide-binding properties of resting and pulsed cytochrome c oxidase in both their stable and transient turnover states. Inhibition of the oxidation of ferrocytochrome c was monitored as a function of cyanide concentration. Cyanide binding to partially reduced forms produced by mixing cytochrome c oxidase with sodium dithionite was also examined. A model is presented that accounts fully for cyanide inhibition of the enzyme, the essential feature of which is the rapid, tight, binding of cyanide to transient, partially reduced, forms of the enzyme populated during turnover. Computer fitting of the experimentally obtained data to the kinetic predictions given by this model indicate that the cyanide-sensitive form of the enzyme binds the ligand with combination constants in excess of 10(6) M-1 X s-1 and with KD values of 50 nM or less. Kinetic difference spectra indicate that cyanide binds to oxidized cytochrome a33+ and that this occurs rapidly only when cytochrome a and CuA are reduced.

The c.d. spectra of Pseudomonas aeruginosa cytochrome c oxidase in the oxidized state and the reduced state are reported in the visible- and u.v. absorption regions. In the visible region the comparison between the spectra of reduced cytochrome c oxidase and ferrocytochrome c-551 allows the identification of the c.d. bands mainly due to the d1 haem chromophore in cytochrome c oxidase. In the near-u.v. region the assignment of some of the observed peaks to the haem groups and to the aromatic amino acid residues is proposed. A careful analysis of the data in the far-u.v. region leads to the determination of the relative amounts of alpha-helix and beta-sheet in the enzyme, giving for the first time a picture of its secondary structure. A significant difference in this respect between the reduced and the oxidized species is observed and discussed in the light of similar conclusions reported by other workers.

The reduction of cytochrome c oxidase (EC 1.9.3.1) by dithionite was investigated by stopped-flow spectrophotometry and flow-flash techniques in the presence of CO. Of the two haem groups present in the enzyme, that associated with cytochrome alpha is the first reduced. The second-order rate constants for reduction of a number of redox proteins (cytochrome c, stellacyanin and azurin) by the S2O4(2-) and SO2.- anions are reported, and the values are compared with those determined for cytochrome c oxidase. These results are discussed in terms of the accessibility and charge distribution of the electron-entry site of cytochrome c oxidase.

Cytochrome c oxidase from ox heart was inserted into artificial liposomal vesicles obtained by sonication of purified soya-bean phospholipids. The cytochrome oxidase vesicles showed a respiratory control ratio of about 2. Spectroscopic properties in the visible and Soret regions and kinetics of CO binding are similar to those of the soluble oxidase. The catalytic efficiency of the cytochrome oxidase vesicles in oxidizing cytochrome c increases as a result of the formation of the ‘pulsed’ form of the oxidase and of the presence in the reaction mixture of carbonyl cyanide p-trifluoromethoxy-phenylhydrazone and nonactin. Analysis of the experimental results obtained under several conditions supports the conclusions that: (i) the alkalinization of the internal microenvironment in the liposomal vesicle is not by itself responsible for the decrease in catalytic activity; (ii) the electrical potential difference created during turnover by proton consumption and/or pumping through the liposome wall is an important mechanism of control in the chain of events leading to the oxidation of external cytochrome c.

Immobilization of biological systems in solid matrices is presently of great interest, in view of the many potential advantages associated with both the higher stability of the immobilized macromolecules and the potential utilization for biotechnology. In the present paper the electrochemical behaviour of the undecapeptide from cytochrome c (called microperoxidase) tightly entrapped in cellulose triacetate membrane is reported; its utilization as ‘solid-state’ promoter in the electrochemistry of soluble metalloproteins is presented. The results obtained indicate that: (i) membrane-entrapped microperoxidase undergoes rapid reversible electron transfer at a glassy carbon electrode; (ii) the electrochemical process is diffusion-controlled; (iii) entrapped microperoxidase acts as ‘solid-state’ promoter in the electrochemistry of soluble cytochrome c and of azurin.

Cytochrome c oxidase was reconstituted in phospholipid vesicles in the presence of highly hydrophobic poly(vinyl alkanoate) polymers. Electron-microscopy observations demonstrated that polymer interaction with the lipid phase induces vesicles to adopt smaller diameters than those typical of standard proteoliposomes. Functional characterization of these polymer-proteoliposome structures indicates that the reconstitution of the enzyme proceeds efficiently without causing either scrambling of the protein orientation in the membrane or loss of respiratory control. A clear dependence of respiratory control ratio on vesicle size was also demonstrated, which is in agreement with a previous model proposed for control of activity of cytochrome c oxidase vesicles [Brunori, Sarti, Colosimo, Antonini, Malatesta, Jones & Wilson (1985) EMBO J. 4, 2365-2368].

Mixing ATP with soluble oxidized cytochrome c oxidase induces a spectral perturbation in the Soret region of the enzyme. This spectral perturbation is observed at ATP concentrations similar to those found to modulate the catalytic activity of cytochrome c oxidase [Malatesta, Antonini, Sarti & Brunori (1987) Biochem. J. 248, 161-165]. The process is reversible and corresponds to a simple binding with Kd = 0.2 mM at 25 degrees C. The absorbance change follows a first-order time course, and analysis of the ATP-concentration-dependence indicates the presence of a rate-limiting monomolecular step that governs the process. From the temperature-dependence of this process, studied at saturating concentrations of ATP, an activation energy of 44 kJ/mol (10.6 kcal/mol) was measured. The spectral perturbation also occurs when cytochrome c oxidase is reconstituted into artificial phospholipid vesicles, with equilibria and kinetics similar to those observed with the soluble enzyme. Mixing ATP with soluble oxidized cyanide-bound cytochrome c oxidase induces a different spectral perturbation, and the apparent affinity of ATP for the enzyme is substantially increased. There is no absolute specificity for ATP, because EGTA, inositol hexakisphosphate, sulphate and phosphate are all able to induce an identical spectral perturbation with the same kinetics, although the value of the apparent Kd is different for the various anions. The presence of Mg2+ ions decreases, in a saturation-dependent fashion, the apparent affinity of cytochrome c oxidase for ATP. The absorbance change can be correlated to the displacement of the Ca2+ bound to cytochrome c oxidase.

Determinations of iron content and dry-weight measurements on samples of Pseudomonas cytochrome oxidase were coupled with sodium dodecyl sulphate/polyacrylamide-gel-electrophoresis studies of both the native protein and covalently cross-linked oligomers in order to estimate the enzyme's molecular weight and spectral absorption coefficients. A value of εox.410=282×103 litre·mol−1·cm−1 was calculated for a dimeric protein molecule having a total molecular weight of 122000 (based on iron analysis). Steady-state kinetic observations of the enzyme-catalysed oxidation of reduced azurin by nitrite indicated a marked increase in enzyme inactivation as the pH was raised from 5.7 to 7.2. Since NO, a product of the nitrite reductase activity of Pseudomonas cytochrome oxidase, is known to bind to the enzyme, a study was undertaken to try to assess the potential of NO as a product inhibitor. Investigations showed that samples of the oxidized protein at pH values 4, 5 and 6 bound NO to both haem c and d1 components, but oxidized enzyme samples at pH7 and above formed their reduced ligand-bound forms when placed under an atmosphere of the gas. Ascorbate-reduced enzyme samples at pH4, 5, 6 and 7 were also found to bind NO at both haem components, although at pH7 the rate of haem c binding was very slow. At pH8 and 9 only the ferrohaem d1 bound NO. Titration experiments on the reduced protein over the pH range 5–7, with nitrite as a precursor of NO, showed that the haem d1 had a much higher affinity than the haem c: experiments at pH5.2 and 5.9 with NO-equilibrated solutions revealed the same pattern of behaviour with the oxidized enzyme.

Ligand-binding equilibria, kinetics and 13C n.m.r. spectra of bound 13CO, of the haemoglobins from two fishes that are very distant on the evolutionary scale, i.e. the fourth haemoglobin component from Salmo irideus and the single component from Osteoglossum bicirrhosum, were studied. The C-terminal sequence was also determined for the haemoglobin from Osteoglossum. The results show that (i) the C-terminal residues of both chains are not directly responsible for the characteristic heterotropic effect known as Root effect, since for both fish haemoglobins these residues are identical with those of human haemoglobins. (ii) In all haemoglobins characterized by the Root effect a dependence of the 13CO n.m.r. resonances on pH is observed. However, the extent of the shift(s) depends on the particular protein, and is probably the result of a combination of both tertiary and quaternary conformational changes. (iii) Both haemoglobins from trout and Osteoglossum manifest a functional heterogeneity between the two types of chains in the tetramer, which increases with proton activity. For CO, the effect is very small for trout haemoglobin IV, and very marked for Osteoglossum haemoglobin; for O2 strongly heterogeneous binding curves were obtained at approx. pH6.2 with both haemoglobins. (iv) Estimations of the relative values of the affinity constants for the α and β chains in the tetramer were obtained for both haemoglobins from 13CO n.m.r. spectra at low fractional saturation. On the basis of these findings the molecular mechanism underlying the Root effect is discussed.

A stopped-flow investigation of the electron-transfer reaction between oxidized azurin and reduced Pseudomonas aeruginosa cytochrome c-551 oxidase and between reduced azurin and oxidized Ps. aeruginosa cytochrome c-551 oxidase was performed. Electrons leave and enter the oxidase molecule via its haem c component, with the oxidation and reduction of the haem d1 occurring by internal electron transfer. The reaction mechanism in both directions is complex. In the direction of oxidase oxidation, two phases assigned on the basis of difference spectra to haem c proceed with rate constants of 3.2 X 10(5)M-1-S-1 and 2.0 X 10(4)M-1-S-1, whereas the haem d1 oxidation occurs at 0.35 +/- 0.1S-1. Addition of CO to the reduced enzyme profoundly modifies the rate of haem c oxidation, with the faster process tending towards a rate limit of 200S-1. Reduction of the oxidase was similarly complex, with a fast haem c phase tending to a rate limit of 120S-1, and a slower phase with a second-order rate of 1.5 X 10(4)M-1-S-1; the internal transfer rate in this direction was o.25 +/- 0.1S-1. These results have been applied to a kinetic model originally developed from temperature-jump studies.

The reduction of cytochrome c oxidase by Cr2+, followed by means of stopped-flow spectrophotometry, exhibits two phases: the faster Cr2+-concentration-dependent reaction has an initial rate constant of 1.1 × 10(4)M-1-S-1, but reaches a rate limit at high concentration of reductant; the slower phase is concentration-independent with a rate of 0.3S-1. The activation energies of the fast and the slow processes are 35 and 71 kJ/mol respectively. The reduction kinetics of the mixed-valence CO complex and the cyanide-inhibited enzyme were compared with those of the fully oxidized forms: both the liganded species have a fast phase identical with that found in the oxidized oxidase. A comparison of the kinetic difference spectra obtained for the fast phase of reduction of oxidized oxidase with those obtained on reduction of the liganded species suggests that the rapid phase arises from the reduction ofhaem a, and the slow phase from the reduction of haem a3.

Horse heart cytochrome c was covalently bound to Sepharose 4B and its redox properties were measured under various experimental conditions. The equilibrium constant for the electron exchange between the oxidized and the reduced form of cytochrome c when one of the two forms was in the semi-solid state and the other one in solution was close to 1. Matrix-bound ferrocytochrome c is very stable to autoxidation and is not oxidized by O2 even in the presence of mammalian cytochrome oxidase. Oxidation occurs if catalytic amounts of soluble cytochrome c are added to the reaction mixture. The rate of oxidation of matrix-bound ferrocytochrome c in the presence of cytochrome oxidase and catalytic amounts of soluble cytochrome c may be correlated with the rate of electron transfer between soluble and matrix-bound cytochrome c. This rate is more than two orders of magnitude lower than that reported for the homonuclear (between identical species) electron transfer in solution.

In stopped-flow experiments in which oxidized cytochrome c oxidase was mixed with ferrocytochrome c in the presence of a range of oxygen concentrations and in the absence and presence of cyanide, a fast phase, reflecting a rapid approach to an equilibrium, was observed. Within this phase, one or two molecules of ferrocytochrome were oxidized per haem group of cytochrome a, depending on the concentration of ferrocytochrome c used. The reasons for this are discussed in terms of a mechanism in which all electrons enter through cytochrome a, which, in turn, is in rapid equilibrium with a second site, identified with ‘visible’ copper (830 nm-absorbing) Cud (Beinert et al., 1971). The value of the bimolecular rate constant for the reaction between cytochromes c2+ and a3+ was between 10(6) and 10(7) M(-1)-S(-1); some variability from preparation to preparation was observed. At high ferrocytochrome c concentrations, the initial reaction of cytochrome c2+ with cytochrome a3+ could be isolated from the reaction involving the ‘visible’ copper and the stoicheiometry was found to approach one molecule of cytochrome c2+ oxidized for each molecule of cytochrome a3+ reduced. At low ferrocytochrome c concentrations, however, both sites (i.e. cytochrome a and Cud) were reduced simultaneously and the stoicheiometry of the initial reaction was closer to two molecules of cytochrome c2+ oxidized per molecule of cytochrome a reduced. The bleaching of the 830 nm band lagged behind or was simultaneous with the formation of the 605 nm band and does not depend on the cytochrome c concentration, whereas the extinction at the steady-state does. The time-course of the return of the 830 nm-absorbing species is much faster than the bleaching of the 605 nm-absorbing component, and parallels that of the turnover phase of cytochrome c2+ oxidation. Additions of cyanide to the oxidase preparations had no effect on the observed stoicheiometry or kinetics of the reduction of cytochrome a and ‘visible’ copper, but inhibited electron transfer to the other two sites, cytochrome a3 and the undetectable copper, Cuu.

The electron-transfer reaction between azurin and cytochrome c1 isolated from Pseudomonas aeruginosa was investigated by rapid-reaction techniques. Temperture-jump studies clearly reveal two chemical relaxations, the amplitudes of which have ikentical spectral distributions, but relaxation times show different dependencies on reactant concentrations. Stopped experiments also showed complex kinetics. A model is proposed which is consistent with the kinetic and equilibrium data obtained. The central feature of this model is the proposal that two intercenvertible forms of reduced azurin exist in solution, only one of which si able to participate directly in the electron-transfer reaction with cytochrome c-551. Support for the hypothesis that two forms of reduced azurin exist is derived from studies on the electron-transfer reaction between azurin and Pseudomonas cytochrome oxidase. The possible physiological significance of such a situation is discussed.

Temperature-jump studies on the electron-transfer reaction between azurin and cytochrome c-551 clearly reveal two chemical relaxations. The amplitudes of these relaxation processes have identical spectral distributions, but the relaxation times show different dependences on the reactant concentrations. These findings are discussed in terms of possible models.

A number of methods were used to prepare a species of mammalian cytochrome oxidase (EC 1.9.3.1, ferrocytochrome c–oxygen oxidoreductase) in which only cytochrome a3 is reduced and in combination with CO. The kinetics of CO binding by cytochrome a32+ in this species is significantly different from that exhibited by cytochrome a32+ in the fully reduced enzyme. The second-order rate constant for combination was 5X104m−1·s−1 and the ‘off’ constant was 3X10−2s−1. The kinetic difference spectra cytochrome a32+–cytochrome a32+–CO reveal further differences between the mixed-valence and the fully reduced enzyme. The reaction between cytochrome a32+ and oxygen in the mixed-valence species was followed in flow–flash experiments and reveals a fast, oxygen-dependent (8X107m−1·s−1 at low oxygen) rate followed by a slow process, whose rate is independent of oxygen but whose amplitude is dependent on [O2]. The fast oxygen-dependent reaction yields as the first product the so-called ‘oxygenated’ enzyme. We conclude from these experiments that the ligand-binding behaviour of cytochrome a3 depends on the redox state of its partners, a fact which represents clear evidence for site–site interaction in this enzyme. The fact that oxygen reacts rapidly with this enzyme species in which only one component, namely cytochrome a3, is reduced represents clear and unequivocal evidence that this is indeed the O2-binding site in cytochrome oxidase and may indicate that reduction of oxygen can proceed via single electron steps.

1. The equilibrium and kinetics of cyanide binding to ferroperoxidase were investigated. At pH9.1 the equilibrium and kinetic measurements agree closely and disclose a single process with an affinity constant of 1.1×103m@!-1 and combination and dissociation velocity constants of 29m-1·s-1 and 2.5×10-2s-1 respectively. 2. At pH values below 8 the affinity constant falls until at pH6.0 the ferroperoxidase·cyanide complex is no longer formed. This is shown to be associated with the formation of ferriperoxidase·cyanide complex in the mixture even in the presence of excess of sodium dithionite. 3. Rapid-pH-jump experiments show a fast pseudo-first-order interconversion between ferroperoxidase·cyanide complex at pH9.1 and ferriperoxidase·cyanide complex at pH6.0. 4. The kinetics of binding of cyanide to dithionite-reduced peroxidase at pH6.0 are complicated and radically different from those observed at pH9.1. 5. Above pH8 the change of affinity constant with pH is consistent with the undissociated species, HCN, being bound by the ferroperoxidase. The enthalpy for this process measured both by equilibrium and kinetic methods is about -8kcal/mol. 6. The binding of cyanide to reconstituted peroxidases, proto, meso and deutero, was investigated. 7. The results are discussed in relation to known data on cyanide binding to other haemoproteins.

The dimeric haemoglobin in the tracheal cells of the Gastrophilus larva was extracted and purified, and the spectral properties of its oxy- and carbon monoxide adducts are recorded. In dilute solutions the kinetic parameters of binding of oxygen and carbon monoxide were determined. In solutions between 0.1 and 50μm for oxygen kon is 1×107m-1·s-1 and koff is 1s-1; for carbon monoxide lon is 6.5×105m-1·s-1 and loff is 0.14s-1. These values are in agreement with previous equilibrium results on oxygen binding and carbon monoxide/oxygen partition. These results are discussed and compared with the known values for other monomeric protohaem proteins.

The equilibrium and kinetics of ethyl isocyanide binding to ferroperoxidase were studied. At pH9.1 the results of both studies are consistent with a single-process model with an affinity constant of 95m−1 and combination and dissociation constants of 2.2×103m−1·s−1 and 23s−1 respectively. Ethyl isocyanide is not bound significantly at pH values lower than 6.0, and in this behaviour and the pH-dependence of the affinity constant, similarities exist between isocyanide and cyanide binding. The enthalpy of the process measured by equilibrium methods is −59kJ/mol (−14kcal/mol). At pH values below 9, the ethyl isocyanide adduct changes in a slow time-dependent manner, giving rise to a new species. These changes are reversible on increasing the pH. The results are discussed in relation to other known information about ligand binding to ferroperoxidase and to myoglobin.

The reaction of Neurospora crassa cytochrome c oxidase with CO was studied by flash-photolysis and rapid-mixing experiments, leading to the determination of the association and dissociation rate constants (7 X 10(4) M-1 X s-1 and 0.02s-1 respectively). Pre-steady-state kinetic investigations of the catalytic properties of the enzyme showed that under proper conditions Neurospora cytochrome c oxidase can be ‘pulsed’, i.e. activated, like the mammalian enzyme. The ‘pulsed’ species is spectroscopically different from the ‘resting’ one, and the decay into the ‘resting’ state is fast (t1/2 approx. 3 min).

The globins from sperm whale and from Aplysia limacina myoglobins were reconstituted by addition of stoichiometric ferric protohaem and the Soret c.d. was followed as a function of time. For both reconstituted proteins, the Soret c.d. changes with time, reflecting haem reorientation inside its pocket, as previously described [Aojula, Wilson & Drake (1986) Biochem. J. 237, 613-616] for sperm whale myoglobin. The time course of the c.d. transition is found to be approx. 10 times faster in Aplysia than in sperm whale myoglobin, a result which is in agreement with the known structural and physicochemical properties of the two myoglobins; furthermore, these results confirm that c.d. and n.m.r. data on haem orientation in haemoproteins reflect the same molecular phenomenon.

The quantum yield, [unk], is reported for the photodissociation of CO from reduced carboxymethylated cytochrome c. The values of [unk] obtained are low relative to that for myoglobin and are pH-independent, being 0.23 at pH6.1 and 0.27 at pH9.7.