The oxygen reactions of reduced cytochrome c oxidase Position of a form with an unusual EPR signal in the sequence of early intermediates

The order of appearance of intermediates in the reoxidation of reduced cytochrome c oxidase by oxygen has been examined. Particular emphasis was placed on determining where the intermediate with the EPR signal at g = 5, 1.78, 1.69 (Shaw, R.W., Hansen, R.E. and Beinert, H. (1978) J. Biol. Chem. 253, 6637–6640) appears in the sequence of events during reoxidation. Flash photolysis of reduced, CO-complexed samples of cytochrome c oxidase in the presence of oxygen in a buffer containing 30% (v/v) ethylene glycol at 77 K and 195 K has been used to generate states of partial reoxidation. The intermediate with the EPR signal at g = 5, 1.78, and 1.69 can be detected as a product of the photolysis and subsequent oxidation but does not appear until the photolyzed sample is incubated at temperatures well above 195 K. In the course of the reoxidation, the intermediate characterized by the g = 5, 1.78, 1.69 signal occurs in the reaction sequence after the states referred to as ‘Compound A’ and ‘Compound B’ (Chance, B., Saronio, C., and Leigh, J.S. (1975) J. Biol. Chem. 250, 9226–9237). Its apperance is within the time range reported for the formation of ‘oxygenated’ cytochrome c oxidase (Orii, Y. (1979) in Cytochrome Oxidase (King. T.E., Orii, Y., Chance, B. and Okunuki, K., eds.), pp. 331–340, Elsevier/North-Holland Biomedical Press, Amsterdam).     

Peroxide interaction with pulsed cytochrome oxidase. Optical and EPR studies

EPR and optical analysis of the 420 nm form of cytochrome oxidase (Kumar, C., Naqui, A., and Chance, B. (1984) J. Biol. Chem. 259, 2073-2076) shows that 1) the 420 nm form possesses a 605 nm band, g = 5 EPR signals, and a slightly blue shifted 655 nm band; 2) the reaction of H2O2 with the 420 nm form generates the peroxide complex (Soret band at 427 nm) with the formation of a 580 nm band and abolition of both the 655 nm band and the g = 5 EPR signal. Comparison of our results with past data shows that various forms of oxidase formed from the resting oxidase through different protocols may be identified to be either the 420 nm or the 427 nm form and leads to identification of a peroxy intermediate during oxidase turnover.     

A novel electron paramagnetic resonance signal of "oxygenated" cytochrome c oxidase.

It had been observed previously that a pair of transient EPR resonances (g = 1.78 and 1.69) appears within less than 5 ms on reoxidation of reduced cytochrome c oxidase by O2. Since the location of other lines that are part of the same signal was not known, the quantity of the paramagnetic species involved, and thus the significance of the observed resonances, remained questionable. We have now found a broad resonance at g = 5 which is obviously associated with those at g = 1.78 and 1.69. The width of the signal (approximately 250 mT) at the observed intensity suggests that it represents a significant fraction of one of the components of the enzyme. The signal disappears within less than 5 ms on addition of cyanide or sulfide but only within several hundred milliseconds after addition of ferrocytochrome c. This behavior suggests that it originates from the a3 component of the enzyme. It is suggested that the species represented in the signal is either identical with or part of what has been named collectively the "oxygenated" form and recently described "activated" forms of the enzyme. On reoxidation of reduced oxidase with oxygen enriched 90% in 17O, no change of signal shape was seen.     

Reactivity of photoreduced cytochrome aa3 complexes with molecular oxygen.

Cytochrome c oxidase (ox heart cytochrome aa3) is reduced on illumination in the presence of a photocatalyst system containing deazaflavin and EDTA. The photo-reduced enzyme reacts with oxygen at neutral pH to give a form of ferric enzyme, whereas a corresponding sample partially reduced by light in the absence of any photocatalyst reacts with oxygen to give an oxyferri species ('oxygenated' enzyme). Reduction by the photocatalyst system at an alkaline pH value (9.0) also gives rise to fully reduced oxidase (both haem groups ferrous). At these pH values the immediate product after oxygen addition is a species with a 605-606 nm absorption band, not identical with ferrous cytochrome a, but capable of oxidizing added cytochrome c. This intermediate, which is unstable at neutral pH, may be analogous to the 'compound B' obtained by Chance and co-workers [Chance, Saronio & Leigh (1975) J. Biol. Chem. 250, 9226-9237; Chance, Saronio & Leigh (1979) Biochem. J. 177, 931-941] at low temperatures.     

Redox-cycled oxidase. One of the reaction products of reduced cytochrome c, cytochrome c oxidase, and dioxygen

Pulsed and oxygenated forms of cytochrome c oxidase are believed to be variants of the oxidized enzyme. They were produced as a consequence of one or more reduction-oxidation cycles of the resting form and are characterized by an increase of the alpha band intensity and a red-shift of the Soret absorption band to 428 nm. The rate of decay of these species back to the resting enzyme varies appreciably and appears to depend on the nature of the reductant and/or oxidant used in their preparation. Here we report that if resting oxidase is incubated with either reduced or oxidized cytochrome c and then exposed to dioxygen, an activated form is rapidly produced which appears to be more oxidized than the starting material. This finding suggest some degree of partial reduction of the resting enzyme, but this by itself cannot explain the extent of activation. Our results further question the significance of the optical spectral "signature" of the oxygenated (Okunuki, K., and Sekuzu, I. (1954) Seitaino Kagaka 5, 265-272), pulsed (Antonini, E., Brunori, M., Colosimo, A., Greenwood, C., and Wilson, M. T. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 3128-3132), and "420 nm" species (Kumar, C., Naqui, A., and Chance, B. (1984) J. Biol. Chem. 259, 2073-2076, 11668-11671), which are thought to be activated forms of oxidized cytochrome c oxidase.     

An infrared study of the binding and photodissociation of carbon monoxide in cytochrome ba3 from Thermus thermophilus

The C-O stretching frequencies of fully reduced carbonmonoxy cytochrome ba3, a newly discovered terminal oxidase of the bacterium Thermus thermophilus (Zimmermann, B.H., Nitsche, C.I., Fee, J.A., Rusnak, F., and Münck, E. (1988) Proc. Natl. Acad. Sci. U.S. A. 85, 5779-5783), are studied by Fourier transform infrared spectroscopy. Multiple C-O frequencies are observed in the Fourier transform infrared spectra, indicating the presence of discrete interconverting conformers of the enzyme. Upon photolysis, the CO is shown to migrate exclusively to CuB+. Above 200 K, the CO returns to the heme a3 by a thermal process which follows simple first-order kinetics. The rate of the reaction was studied from 205 to 230 K and at 300 K, yielding the activation parameters delta H = 14.9 kcal/mol and delta S = -5 cal/mol/K. These are compared with previously determined activation parameters for CO recombination in mitochondrial cytochrome aa3 preparations (Fiamingo, F.G., Altschuld, R.A., Moh, P.P., and Alben, J.O. (1982) J. Biol. Chem. 257, 1639-1650). We report the novel finding that CO remains bound to CuB+ at room temperature during continuous photolysis of cytochrome ba3, and we conjecture on the possible interference of copper-bound CO in "flow-flash" and "triple-trap" studies of cytochrome c oxidases.     

Cytochrome c oxidase from bakers' yeast. V. Arrangement of the subunits in the isolated and membrane-bound enzyme.

Cytochrome c oxidase from baker's yeast contains three mitochondrially made subunits (I to III) which are relatively hydrophobic and four cytoplasmically made subunits (IV to VII) which are relatively hydrophilic (Mason, T. L., Poyton, R. O., Wharton, D.C., and Schatz, G. (1973) J. Biol. Chem. 248, 1346-1354 and Poyton, R. O., and Schatz, G. (1975) J. Biol. Chem. 250, 752-761). In order to explore the arrangement of these subunits in the holoenzyme, the reactivity of each subunit with a variety of "surface probes" was tested with isolated cytochrome c oxidase, with cytochrome c oxidase incorporated into liposomes, and with mitochondrially bound cytochrome c oxidase. The surface probes included iodination with lactoperoxidase and coupling with p-diazonium benzenesulfonate. In addition, external subunits were identified by linking them to bovine serum albumin carrying a covalently bound isocyanate group. In the membrane-bound enzyme, Subunit I was almost completely inaccessible and Subunit II was partly inaccessible to all surface probes. All of the other subunits were accessible. Similar results were obtained with the solubilized enzyme, except that the differences in reactivity between the individual subunits were less clear-cut. The results obtained with liposome-bound cytochrome c oxidase resembled those obtained with the mitochondrially bound enzyme. These data suggest that the two largest mitochondrially made subunits are localized in the interior of the enzyme and that they are genuine components of cytochrome c oxidase.     

Binding of arylazidocytochrome c derivatives to beef heart cytochrome c oxidase: cross-linking in the high- and low-affinity binding sites

Two arylazidocytochrome c derivatives, one modified at lysine-13 and the second modified at lysine-22, were reacted with beef heart cytochrome c oxidase. The lysine-13 modified arylazidocytochrome c was found to cross-link both to the enzyme and with lipid bound to the cytochrome c oxidase complex. The lysine-22 derivative reacted only with lipids. Cross-linking to protein was through subunit II of the cytochrome c oxidase complex, as first reported by Bisson et al. [Bisson, R., Azzi, A., Gutweniger, H., Colonna, R., Monteccuco, C., & Zanotti, A. (1978) J. Biol. Chem. 253, 1874]. Binding studies show that the cytochrome c derivative covalently bound to subunit II was in the high-affinity binding site for the substrate. Evidence is also presented to suggest that cytochrome c bound to the lipid was in the low-affinity binding site [as defined by Ferguson-Miller et al. [Ferguson-Miller, S., Brautigan, D. L., & Margoliash, E. (1976) J. Biol. Chem. 251, 1104]]. Covalent binding of the cytochrome c derivative into the high-affinity binding site was found to inhibit electron transfer even when native cytochrome c was added as a substrate. Inhibition was almost complete when 1 mol of the Lys-13 modified arylazidocytochrome c was covalently bound to the enzyme per cytochrome c oxidase dimer (i.e., congruent to 280 000 daltons). Covalent binding of either derivative with lipid (low-affinity site) had very little effect on the overall electron transfer activity of cytochrome c oxidase. These results are discussed in terms of current theories of cytochrome c-cytochrome c oxidase interactions.     

Presence of coupled trinuclear copper cluster in mammalian ceruloplasmin is essential for efficient electron transfer to oxygen

The reactivity with dioxygen of a mammalian (sheep) ceruloplasmin, anaerobically reduced with ascorbate, was found to depend on the state of the Type 2 and Type 3 copper centers, as monitored by EPR and optical spectroscopy. A complete reoxidation by air after anaerobic reduction with ascorbate was observed with samples (A) purified by the single-step procedure described for chicken ceruloplasmin (Calabrese, L., Carbonaro, M., and Musci, G. (1988) J. Biol. Chem. 263, 6480-6483), while samples prepared by traditional multistep procedure (B) or subjected to freeze-thawing (C) displayed partial and very slow reoxidation, reflecting the functional nonequivalence of blue coppers which is considered a typical property of mammalian ceruloplasmin. The rate of reduction of the 330 nm chromophore was found to increase as a function of the extent and rate of reoxidation of different samples, while the 610 nm band displayed an opposite trend. Samples B and C showed a Type 2 copper signal in the EPR spectrum, while sample A showed practically no Type 2 copper in the oxidized protein, and a transient Type 2-like signal during reduction. The presence of a trinuclear Type 2-Type 3 cluster can therefore be proposed for all ceruloplasmins, and the integrity of the copper-copper coupling is essential for efficient oxidase behavior.     

Adrenodoxin reductase and adrenodoxin. Mechanisms of reduction of ferricyanide and cytochrome c.

Adrenodoxin reductase, the flavoprotein moiety of the adrenal cortex mitochondrial steroid hydroxylating system, participates in adrenodoxin-dependent cytochrome c and adrenodoxin-independent ferricyanide reduction, with NADPH as electron donor for both of these 1-electron reductions. For ferricyanide reduction, adrenodoxin reductase cycles between oxidized and 2-electron-reduced forms, reoxidation proceeding via the neutral flavin (FAD) semiquinone form (Fig. 9). Addition of adrenodoxin has no effect upon the kinetic parameters of flavoprotein-catalyzed ferricyanide reduction. For cytochrome c reduction, the adrenodoxin reductase-adrenodoxin 1:1 complex has been shown to be the catalytically active species (Lambeth, J. D., McCaslin, D. R., and Kamin, H. (1976) J. Biol. Chem. 251, 7545-7550). Present studies, using stopped flow techniques, have shown that the 2-electron-reduced form of the complex (produced by reaction with 1 eq of NADPH) reacts rapidly with 1 eq of cytochrome c (k approximately or equal to 4.6 s-1), but only slowly with a second cytochrome c (k = 0.1 to 0.3 s-1). However, when a second NADPH is included, two more equivalents of cytochrome are reduced rapidly. Thus, the adrenodoxin reductase-adrenodoxin complex appears to cycle between 1- and 3-electron reduced states, via an intermediate 2-electron-containing form produced by reoxidation by cytochrome (Fig. 10). For ferricyanide reduction by adrenodoxin reductase, the fully reduced and semiquinone forms of flavin each transfer 1 electron at oxidation-reduction potentials which differ by approximately 130 mV. However, adrenodoxin in a complex with adrenodoxin reductase allows electrons of constant potential to be delivered from flavin to cytochrome c via the iron sulfur center...     

Definition of the interaction domain for cytochrome c on cytochrome c oxidase. Ii. Rapid kinetic analysis of electron transfer from cytochrome c to Rhodobacter sphaeroides cytochrome oxidase surface mutants

The reaction between cytochrome c (Cc) and Rhodobacter sphaeroides cytochrome c oxidase (CcO) was studied using a cytochrome c derivative labeled with ruthenium trisbipyridine at lysine 55 (Ru-55-Cc). Flash photolysis of a 1:1 complex between Ru-55-Cc and CcO at low ionic strength results in electron transfer from photoreduced heme c to Cu(A) with an intracomplex rate constant of k(a) = 4 x 10(4) s(-1), followed by electron transfer from Cu(A) to heme a with a rate constant of k(b) = 9 x 10(4) s(-1). The effects of CcO surface mutations on the kinetics follow the order D214N > E157Q > E148Q > D195N > D151N/E152Q approximately D188N/E189Q approximately wild type, indicating that the acidic residues Asp(214), Glu(157), Glu(148), and Asp(195) on subunit II interact electrostatically with the lysines surrounding the heme crevice of Cc. Mutating the highly conserved tryptophan residue, Trp(143), to Phe or Ala decreased the intracomplex electron transfer rate constant k(a) by 450- and 1200-fold, respectively, without affecting the dissociation constant K(D). It therefore appears that the indole ring of Trp(143) mediates electron transfer from the heme group of Cc to Cu(A). These results are consistent with steady-state kinetic results (Zhen, Y., Hoganson, C. W., Babcock, G. T., and Ferguson-Miller, S. (1999) J. Biol. Chem. 274, 38032-38041) and a computational docking analysis (Roberts, V. A., and Pique, M. E. (1999) J. Biol. Chem. 274, 38051-38060).     

Kinetic studies on cytochrome c oxidase by combined epr and reflectance spectroscopy after rapid freezing.

1. Techniques and experiments are described concerned with the millisecond kinetics of EPT-detectable changes brought about in cytochrome c oxidase by reduced cytochrome c and, after reduction with various agents, by reoxidation with O2 or ferricyanide. Some experiments in the presence of ligands are also reported. Light absorption was monitored by low-temperature reflectance spectroscopy. 2. In the rapid phase of reduction of cytochrome c oxidase by cytochrome c (less than 50 ms) approx. 0.5 electron equivalent per heme a is transferred mainly to the low-spin heme component of cytochrome c oxidase and partly to the EPR-detectable copper. In a slow phase (less than 1 s) the copper is reoxidized and high-spin ferric heme signals appear with a predominant rhombic component. Simultaneously the absorption band at 655 nm decreases and the Soret band at 444 nm appears between the split Soret band (442 and 447 nm) of reduced cytochrome a. 3. On reoxidation of reduced enzyme by oxygen all EPR and optical features are restored within 6 ms. On reoxidation by O2 in the presence of an excess of reduced cytochrome c, states can be observed where the low-spin heme and copper signals are largely absent but the absorption at 655 nm is maximal, indicating that the low-spin heme and copper components are at the substrate side and the component(s) represented in the 655 nm absorption at the O2 side of the system. On reoxidation with ferricyanide the 655 nm absorption is not readily restored but a ferric high-spin heme, represented by a strong rhombic signal, accumulates. 4. On reoxidation of partly reduced enzyme by oxygen, the rhombic high-spin signals disappear within 6 ms., whereas the axial signals disappear more slowly, indicating that these species are not in rapid equilibrium. Similar observations are made when partly reduced enzyme is mixed with CO. 5. The results of this and the accompanying paper are discussed and on this basis an assignment of the major EPR signals and of the 655 nm absorption is proposed, which in essence is that published previously (Hartzell, C.R., Hansen, R.E. and Beinert, H. (1973) Proc. Natl. Acad. Sci. U.S. 70, 2477-2481). Both the low-spin (g=o; 2.2; 1.5) and slowly appearing high-spin (g=6; 2) signals are attributed to ferric cytochrome a, whereas the 655 nm absorption is thought to arise from ferric cytochrome a3, when it is present in a state of interaction with EPR-undectectable copper. Alternative possibilities and possible inconsistencies with this proposal are discussed.     

An investigation by e.p.r. and optical spectroscopy of cytochrome oxidase during turnover.

Cytochrome oxidase (EC 1.9.3.1; ferrocytochrome c:oxygen oxidoreductase) was studied during steady-state by optical and e.p.r. methods. Starting with either the 'resting' or the 'pulsed' enzyme, oxidase, cytochrome c, ascorbate and O2 were mixed and the reaction monitored optically. Tetramethylphenylenediamine was used as mediator to poise the steady-state to the desired reduction level. After mixing, the reaction was quenched by the used of rapid-freeze techniques. The e.p.r. spectra of samples captured at increasing tetramethylphenylenediamine concentrations (i.e. higher electron flux) show decreasing g = 2 (Cu A) and g = 3 (cytochrome a) signals. No Cu B or g = 6 signals (high-spin cytochrome a3) could be found during the reaction. Also, the signal with peaks at g = 1.69, 1.78 and 5 as well as the g = 12 signal was hardly detectable at higher turnover rates. The only new signal appearing during turnover is a radical signal, which is discussed in terms of a protein radical. Finally, a scheme is presented, proposing a catalytic cycle for cytochrome oxidase with respect to the O2 binding Cu B-cytochrome a3 unit.     

Kinetics and energetics of intramolecular electron transfer in yeast cytochrome c peroxidase

The oxidation of ferric cytochrome c peroxidase by hydrogen peroxide yields a product, compound ES [Yonetani, T., Schleyer, H., Chance, B., & Ehrenberg, A. (1967) in Hemes and Hemoproteins (Chance, B., Estabrook, R. W., & Yonetani, T., Eds.) p 293, Academic Press, New York], containing an oxyferryl heme and a protein free radical [Dolphin, D., Forman, A., Borg, D. C., Fajer, J., & Felton, R. H. (1971) Proc. Natl. Acad. Sci. U.S.A. 68, 614-618]. The same oxidant takes the ferrous form of the enzyme to a stable Fe(IV) peroxidase [Ho, P. S., Hoffman, B. M., Kang, C. H., & Margoliash, E. (1983) J. Biol. Chem. 258, 4356-4363]. It is 1 equiv more highly oxidized than the ferric protein, contains the oxyferryl heme, but leaves the radical site unoxidized. Addition of sodium fluoride to Fe(IV) peroxidase gives a product with an optical spectrum similar to that of the fluoride complex of the ferric enzyme. However, reductive titration and electron paramagnetic resonance (EPR) data demonstrate that the oxidizing equivalent has not been lost but rather transferred to the radical site. The EPR spectrum for the radical species in the presence of Fe(III) heme is identical with that of compound ES, indicating that the unusual characteristics of the radical EPR signal do not result from coupling to the heme site. By stopped-flow measurements, the oxidizing equivalent transfer process between heme and radical site is first order, with a rate constant of 0.115 s-1 at room temperature, which is independent of either ligand or protein concentration.(ABSTRACT TRUNCATED AT 250 WORDS)     

The identity of a new copper(II) electron paramagnetic resonance signal in cytochrome c oxidase

In reoxidation experiments with cytochrome c oxidase (EC 1.9.3.1) in the presence of both reducing substrate and molecular oxygen, a new EPR signal from Cu2+ has been observed. The new signal corresponds to 0.45 Cu per functional unit. It is concluded that the new EPR signal originates from CuB2+, the copper which is EPR-nondetectable in the resting enzyme. Optical absorption changes in the 500-700 nm region accompanies the decay of the new Cu2+ EPR signal. Based on the results in this investigation a catalytic cycle for cytochrome oxidase is proposed.     

Sulfite oxidase from chicken liver. The role of imidazole and carboxyl groups for the reaction with cytochrome c

Oxidation of sulfite to sulfate by sulfite oxidase is inhibited when the enzyme is treated with reagents known to modify imidazole and carboxyl groups. Modification inhibits the oxidation of sulfite by the physiological electron acceptor cytochrome c, but not by the artificial acceptor ferricyanide. This indicates interference with reaction steps that follow the oxidation of sulfite by the enzyme's molybdenum cofactor. Reaction with diethylpyrocarbonate modifies ten histidines per enzyme monomer. Loss of activity is concomitant to the modification of only a single histidine residue. Inactivation takes place at the same rate in free sulfite oxidase and in the sulfite-oxidase--cytochrome-c complex. Blocking of carboxyl groups with water-soluble carbodiimides inactivates the enzyme. But none of the enzyme's carboxyl groups seems to be essential in the sense that its modification fully abolishes activity. The pattern of inactivation by chemical modification of sulfite oxidase is quite similar to that observed previously for cytochrome c peroxidase from yeast [Bosshard, H. R., B?nziger, J., Hasler, T. and Poulos, T. L. (1984) J. Biol. Chem. 259, 5683-5690; Bechtold, R. and Bosshard, H. R. (1985) J. Biol. Chem. 260, 5191-5200]. The two enzymes have very different structures yet share cytochrome c as a common substrate of which they recognize the same electron-transfer domain around the exposed heme edge.     

Definition of the interaction domain for cytochrome c on cytochrome c oxidase. I. Biochemical, spectral, and kinetic characterization of surface mutants in subunit ii of Rhodobacter sphaeroides cytochrome aa(3)

To determine the interaction site for cytochrome c (Cc) on cytochrome c oxidase (CcO), a number of conserved carboxyl residues in subunit II of Rhodobacter sphaeroides CcO were mutated to neutral forms. A highly conserved tryptophan, Trp(143), was also mutated to phenylalanine and alanine. Spectroscopic and metal analyses of the surface carboxyl mutants revealed no overall structural changes. The double mutants D188Q/E189N and D151Q/E152N exhibit similar steady-state kinetic behavior as wild-type oxidase with horse Cc and R. sphaeroides Cc(2), showing that these residues are not involved in Cc binding. The single mutants E148Q, E157Q, D195N, and D214N have decreased activities and increased K(m) values, indicating they contribute to the Cc:CcO interface. However, their reactions with horse and R. sphaeroides Cc are different, as expected from the different distribution of surface lysines on these cytochromes c. Mutations at Trp(143) severely inhibit activity without changing the K(m) for Cc or disturbing the adjacent Cu(A) center. From these data, we identify a Cc binding area on CcO with Trp(143) and Asp(214) close to the site of electron transfer and Glu(148), Glu(157), and Asp(195) providing electrostatic guidance. The results are completely consistent with time-resolved kinetic measurements (Wang, K., Zhen, Y., Sadoski, R., Grinnell, S., Geren, L., Ferguson-Miller, S., Durham, B., and Millett, F. (1999) J. Biol. Chem. 274, 38042-38050) and computational docking analysis (Roberts, V. A., and Pique, M. E. (1999) J. Biol. Chem. 274, 38051-38060).     

Studies of the ferredoxin from Thermus thermophilus

The soluble ferredoxin from Thermus thermophilus was examined by M?ssbauer and EPR spectroscopies and by reductive titrations. These studies demonstrate the presence of one 3Fe center, responsible for the characteristic g = 2.02 EPR signal in the oxidized protein, and one [4Fe-4S] center which is responsible for the rhombic EPR spectrum of the fully reduced protein. These assignments should replace those made by Ohnishi et al. (Ohnishi, T., Blum, H., Sato, S., Nakazawa, K., Hon-nami, K., and Oshima, T. (1980) J. Biol. Chem. 255, 345-348) prior to the discovery of the 3Fe clusters. The amino acid composition was determined and is discussed with reference to recent structural studies of 7Fe ferredoxins.     

A re-evaluation of the low-temperature kinetics of the reaction of fully reduced mitochondrial cytochrome oxidase with carbon monoxide and the spectral characterization of species Ic in the Soret and visible regions

The kinetics of the reaction of fully reduced membrane bound cytochrome oxidase with CO following photolysis of the fully reduced cytochrome oxidase-CO complex habe been re-examined by re-analysing the data of Clore and Chance (1978) Biochem. J. 175, 709-725) at six temperatures in the 178-203 K range simultaneously at only a single wavelength pair, 444-463 nm. The choice of the 444-463 nm wavelength pair was based on the fact that the absorbance change produced at 444-463 nm on photolysis of the CO complex is sufficiently large and the separation between monitoring and reference wavelengths sufficiently small to render the effects of any possible time dependent scattering changes insignificant. On the basis of our analysis only a two step mechanism (Model 1 of Clore and Chance (1978) Biochem. J. 175, 709-725) satisfies the triple requirement of a S.D. within the standard error of the data, a random distribution of residuals and good determination of the optimized parameters. The single step mechanism of De Fonseka and Chance (1978) Biochem. J. 175, 1137-1138) fails to satisfy all three requirements. The pure difference spectra of species Ic minus E, E minus IIc and Ic minus IIc are calculated from the computed kinetics of the individual species and repetitive slow wavelength scanning difference spectra (reaction sample minus the CO complex) taken during the course of the reaction of fully reduced cytochrome oxidase with CO at 176 K.     

Electron paramagnetic resonance of the tungsten derivative of rat liver sulfite oxidase.

Sulfite oxidase purified from livers of tungsten-treated rats has been used for EPR studies of tungsten substituted at the molybdenum site of the enzyme in a fraction of the molecules. The EPR signal of W(V) in sulfite oxidase is quite similar to that of Mo(V) in its line shape and in its sensitivity to the presence of anions such as phosphate and fluoride. Hyperfine interaction with a dissociable proton is also observed in both signals. The pH-dependent alteration in line shape exhibited by the Mo(V) EPR signal of the rat liver enzyme. Incomplete reduction of the tungsten center at pH 9 is indicated by attenuated signal intensity at this pH. The W(V) signal has g values lower than those of the Mo(V) signal, has a much broader resonance envelope, and is much less readily saturated by increasing microwave power. Kinetic studies on the reduction of the heme and tungsten centers of sulfite oxidase have shown that reduction of de-molybdo forms of sulfite oxidase by sulfite is catalyzed by the residual traces of native molybdenum-containing molecules. Reduction is accomplished by electron transfer involving intermolecular heme-heme interaction. The W(V) signal is generated only after all the heme centers are reduced. The rate and extent of heme reduction at pH 9 are the same as at pH 7. Studies on the reoxidation of W(V) and reduced heme by O2 and by cytochrome c suggest that the cytochrome b5 of sulfite oxidase is the site of electron transfer to cytochrome c, whereas oxidase activity is the property of the molybdenum center. It appears that the tungsten center in sulfite oxidase is incapable of oxidizing sulfite.