Impaired mitochondrial beta-oxidation in a patient with an abnormality of the respiratory chain. Studies in skeletal muscle mitochondria.

Defects of complex I of the mitochondrial respiratory chain are important causes of neurological disease. We report studies that demonstrate a severe deficiency of complex I activity with less severe abnormalities of complexes III and IV (less than 5, 63, and 30% of control values, respectively) in a skeletal muscle mitochondrial fraction from a 22-yr-old female with weakness, lactic acidemia, and the deposition of intramuscular neutral lipid. The observation that lipid accumulates in this and other patients with complex I deficiency suggests impaired mitochondrial fatty acid oxidation. To investigate this mechanism we have shown impaired flux through beta-oxidation [( U-14C]hexadecanoate oxidation was 66% of control rate) and accumulation of specific acyl-CoA ester intermediates. The changes in fatty acid metabolism in complex I deficiency are secondary to the reduced state within the mitochondrial matrix with low NAD+/NADH ratios.

Inorganic nitrogen metabolism in bacteria.

Enzymatic reactions involving inorganic nitrogen species provide a rich variety of systems with which to study biological chemistry. In many cases, catalysis involves redox chemistry and takes place at metal centres. Recent structures and new spectroscopic data have rapidly advanced our knowledge of nitrogen cycle enzymology, particularly in the areas of nitrogen fixation, hydroxylamine oxidation and nitrite reduction. In the case of the nitrate reductases and nitric oxide reductase, models for structure and catalysis can be designed, based on new structural information that is now available for closely related enzymes. The past two years have also seen significant progress in our understanding of the enzymology of some 'new' reactions of the nitrogen cycle, for example anaerobic ammona oxidation and heterotrophic nitrification.

The reaction of Escherichia coli cytochrome bo with H2O2: evidence for the formation of an oxyferryl species by two distinct routes.

We have re-examined the reaction of fast oxidised cytochrome bo with H202 in a stopped-flow spectrophotometer. Monitoring the reaction at 582 nm allows us to observe the formation and decay of a spectroscopically distinct intermediate which accumulates transiently prior to the formation of an oxyferryl species previously characterised in this laboratory (Watmough, N.J., Cheesman, M.R., Greenwood, C. and Thomson, A.J. (1994) Biochem. J. 300, 469-475 [1]). The reaction shows three distinct phases of which the fast and intermediate phases are bimolecular and show a marked pH dependence. Initially these results appeared incompatible with the report that only one equivalent of H202 is required to generate the oxyferryl species (Moody, A.J. and Rich, P.R. (1994) Eur. J. Biochem. 226, 731-737 [21]. However, these data can be reconciled by a branched reaction mechanism whose contributions differ according to the peroxide concentration used.

Distinct forms of the haem o-Cu binuclear site of oxidised cytochrome bo from Escherichia coli. Evidence from optical and EPR spectroscopy.

Oxidised, formate-bound and fluoride-bound forms of E. coli cytochrome bo give rise to an electronic absorption band near 630 nm, diagnostic of high-spin ferric haem o, whose position is sensitive to the nature of the bound anion. In all three forms, haem o remains spin-coupled to Cu(B)(II), resulting in distinct broad X-band EPR signals. Those of formate-bound cytochrome bo are similar to the signals seen in slow cytochrome aa3 but cannot be induced by incubation at acid pH suggesting that the endogenous carboxylate believed to be important in slow cytochrome aa3 is not present in cytochrome bo. The oxidised form gives rise to novel EPR signals at g = 3.74 and g = 3.08 which have not been detected in cytochrome aa3 and may arise from a weak magnetic coupling between high-spin haem o, S = 5/2, and Cu(B)(II), S = 1/2.

Effects of tenotomy on muscle histology and energy metabolism in normal and dystrophic mice.

Creatine, phosphocreatine, ATP and phosphate concentrations were measured in extracts of tibialis anterior and gastrocnemius muscles from 42 normal and 39 dystrophic mice from 1 to 16 weeks of age. No differences were observed at 1 week, prior to the onset of histological abnormalities in dystrophic animals. Creatine and phosphocreatine concentrations were significantly reduced in older dystrophic mice, and phosphate levels were higher, while ATP levels generally did not differ. Tenotomy of gastrocnemius at 1 week prevented the development of dystrophy in this muscle but this was not associated with an increase in phosphogen concentrations. Serum creatine kinase levels were significantly higher in dystrophic mice than normal mice but only during the first two weeks of life; levels in older mice were not significantly different. This study shows that the reported deficits in phosphogen concentrations in dystrophic muscles are likely to reflect the results, rather than the cause, of the dystrophic process.

A new simple screening method for the diagnosis of medium chain acyl-CoA dehydrogenase deficiency.

We describe a simple inexpensive method for the detection of octanoyl-carnitine in urine by reverse-phase high performance thin-layer chromatography of the p-bromophenacyl derivative. This method provides a rapid and reliable means for the diagnosis of medium-chain acyl-CoA dehydrogenase deficiency which is suitable for routine laboratory use.

Measurement of phosphate concentration in the presence of very labile phosphate esters.

There are a number of methods available for the measurement of phosphate ion concentration, which may be used when moderately labile phosphate esters such as ATP are present in low concentration. However, the highly acidic conditions usually employed make these unsuitable when very labile esters such as phosphocreatine are present. A method in which the phosphomolybdate complex is developed under mildly acidic conditions, using high molybdate concentrations to counteract the reduced assay sensitivity at high pH, is described. The assay is linear in the range 5-300 microM phosphate, and micromolar concentrations of phosphate can be reliably measured in the presence of millimolar phosphocreatine.

A switch in heme axial ligation prepares Paracoccus pantotrophus cytochrome cd1 for catalysis.

Cytochrome cd1 nitrite reductase (cd1) from Paracoccus pantotrophus is a respiratory enzyme capable of using nitrite, hydroxylamine and oxygen as electron accepting substrates. Structural studies have shown that when the enzyme is reduced there is a change in the axial ligation of both hemes, which has been proposed to form part of the catalytic cycle. Here we report the use of a physiological electron donor, pseudoazurin, to investigate the relationship between heme ligation and catalysis. A combination of visible absorption and electron paramagnetic resonance spectroscopies reveals the formation of a catalytically competent state of oxidized cd1 with 'switched' axial ligands immediately after complete reoxidation of reduced cd1 with hydroxylamine. This activated conformer returns over 20 min at 25 degrees C to the state previously observed for oxidized 'as isolated' cd1, which is catalytically inactive towards the same substrates.

Electron transfer between cytochrome c and the isolated CuA domain: identification of substrate-binding residues in cytochrome c oxidase.

Subunit II of cytochrome c oxidase has a C-terminal domain that is exposed to aqueous solution on membrane surface and contains a copper center called CuA. The central part of the cytochrome c binding site is thought to reside in this domain. We have expressed the subunit II fragment of the Paracoccus denitrificans cytochrome c oxidase in a soluble form and studied its interaction with cytochrome c by stopped-flow spectroscopy. The oxidation of cytochrome c by the CuA domain follows monophasic kinetics, indicating the presence of a single kinetically competent binding site. In low ionic strength medium, the domain oxidizes Paracoccus cytochrome c-550 and horse mitochondrial cytochrome c at the rates of 1.5 x 10(6) and 3 x 10(5) M-1 s-1, respectively. The reaction rates are strongly dependent on ionic strength, which must reflect electrostatic interactions within the complex. The KD for the complex between the bacterial cytochrome c and the domain is 1.6 microM; i.e., it is similar to that between the mitochondrial cytochrome c and the intact oxidase, suggesting that both contain the same catalytically competent binding site. Using site-directed mutagenesis, we have identified five conserved residues of the CuA domain that are involved in the cytochrome c binding. Mutations of glutamine 148, glutamate 154, aspartate 206, aspartate 221, or glutamate 246 lead to a 35-85% decrease in the rate of cytochrome c oxidation. The simultaneous substitution of three invariant carboxylic acids (aspartate 206, aspartate 221, and glutamate 246) leads to a 95% decrease in the reaction rate. Conversely, the reaction can be enhanced by removing a positive charge (lysine 219) from the CuA domain.

Structural and redox relationships between Paracoccus denitrificans, porcine and human electron-transferring flavoproteins.

Electron-transferring flavoprotein (ETF) was purified from the bacterium Paracoccus denitrificans and the structural and redox relationships to the porcine and human ETFs were investigated. The three proteins have essentially identical subunit masses and the alpha-helix content of the bacterial and porcine ETFs are very similar, indicating global structural similarity. An anti-(porcine ETF) polyclonal antibody that crossreacts with the human large and small subunits also crossreacts strongly with the large subunit of Paracoccus ETF. However, crossreactivity with the small subunit is very weak. Nonetheless, an amino-terminal peptide and four internal peptides of the small bacterial subunit show extensive sequence identity with the human small subunit. Local similarities in environment are also indicated by the intrinsic tryptophan fluorescence emission spectra of porcine and Paracoccus ETFs. Although the visible spectra of porcine and Paracoccus ETFs are virtually identical, flavin fluorescence in the bacterial protein is only 15% that of the mammalian protein. Further, the circular dichroic spectrum of the flavin in the bacterial protein is significantly more intense, suggesting that the microenvironment of the isoalloxazine ring is different in the two proteins. Enzymatic or photochemical reduction of Paracoccus ETF rapidly yields an anionic semiquinone; formation of the fully reduced flavin in the bacterial ETF is very slow. The spacing of the oxidation-reduction potentials of the flavin couples in the bacterial ETF is essentially identical to that in procine ETF as judged from the disproportionation equilibrium of the bacterial ETF flavin semiquinone. Together, the enzymatic reduction and disproportionation equilibria suggest that the flavin potentials of the two ETFs must be very close. The data indicate that the structural properties of the bacterial and mammalian proteins and the thermodynamic properties of the flavin prosthetic group of the proteins are very similar.

Cytochrome cbb(3) oxidase and bacterial microaerobic metabolism.

Cytochrome cbb(3) oxidase is a member of the haem-copper oxidase superfamily. It is characterized by its high oxygen affinity, while retaining the ability to pump protons. These attributes are central to its proposed role in bacterial microaerobic metabolism. Recent spectroscopic characterization of both the cytochrome cbb(3) oxidase complex from Pseudomonas stutzeri and the dihaem ccoP subunit expressed separately in Escherichia coli has revealed the presence of a low-spin His/His co-ordinated c-type cytochrome. The low midpoint reduction potential of this haem (E(m)<+100 mV), together with its unexpected ability to bind CO in the reduced state at the expense of the distal histidine ligand, raises questions about the role of the ccoP subunit in the delivery of electrons to the active site.

Two conserved glutamates in the bacterial nitric oxide reductase are essential for activity but not assembly of the enzyme.

The bacterial nitric oxide reductase (NOR) is a divergent member of the family of respiratory heme-copper oxidases. It differs from other family members in that it contains an Fe(B)-heme-Fe dinuclear catalytic center rather than a Cu(B)-heme-Fe center and in that it does not pump protons. Several glutamate residues are conserved in NORs but are absent in other heme-copper oxidases. To facilitate mutagenesis-based studies of these residues in Paracoccus denitrificans NOR, we developed two expression systems that enable inactive or poorly active NOR to be expressed, characterized in vivo, and purified. These are (i) a homologous system utilizing the cycA promoter to drive aerobic expression of NOR in P. denitrificans and (ii) a heterologous system which provides the first example of the expression of an integral-membrane cytochrome bc complex in Escherichia coli. Alanine substitutions for three of the conserved glutamate residues (E125, E198, and E202) were introduced into NOR, and the proteins were expressed in P. denitrificans and E. coli. Characterization in intact cells and membranes has demonstrated that two of the glutamates are essential for normal levels of NOR activity: E125, which is predicted to be on the periplasmic surface close to helix IV, and E198, which is predicted to lie in the middle of transmembrane helix VI. The subsequent purification and spectroscopic characterization of these enzymes established that they are stable and have a wild-type cofactor composition. Possible roles for these glutamates in proton uptake and the chemistry of NO reduction at the active site are discussed.

Cytochrome bo from Escherichia coli: reaction of the oxidized enzyme with hydrogen peroxide.

Oxidized cytochrome bo reacts rapidly with micromolar concentrations of H2O2 to form a single derivative. The electronic absorption spectrum of this compound differs from that of the oxidized form of the enzyme reported by this laboratory [Watmough, Cheesman, Gennis, Greenwood and Thomson (1993) FEBS Lett. 319, 151-154]. It is characterized by a Soret maximum at 411 nm, increased absorbance at 555 nm, and reduced intensity at 624 nm. The apparent dissociation constant for this process is of the order of 4 x 10(-6) M, and the bimolecular rate constant for the formation of the new compound is (1.25-1.7) x 10(3) M-1.s-1. Electronic absorption difference spectroscopy shows this product to be identical with the compound formed from the reaction of the mixed-valence form of the enzyme with dioxygen. Investigation of this compound by room-temperature magnetic c.d. spectroscopy shows haem o to be neither high-spin nor low-spin ferric, but to have a spectrum characteristic of an oxyferryl species. There is no evidence for oxidation of the porphyrin ring. Therefore the binuclear centre of this species must consist of an oxyferryl haem (S = 1) coupled to a Cu(II) ion (S = 1/2) to form a new paramagnetic centre. The reaction was also followed by X-band e.p.r. spectroscopy, and this showed the disappearance in parallel with the formation of the oxyferryl species, of the broad g = 3.7, signal which arises from the weakly coupled binuclear centre in the oxidized enzyme. Since no new e.p.r.-detectable paramagnetic species were observed, the Cu(II) ion is presumed to be coupled to another paramagnet, possibly an organic radical. There is no evidence in the electronic absorption spectrum to indicate further reaction of cytochrome bo with H2O2 to form a second species. We argue that the circumstances of formation of this oxyferryl species are the same as those for the P form of cytochrome c oxidase, a species often regarded as containing a bound peroxide ion. The implications of these observations for the reaction mechanism of haem-copper terminal oxidases are discussed.

Tryptophan fluorescence in electron-transfer flavoprotein:ubiquinone oxidoreductase: fluorescence quenching by a brominated pseudosubstrate.

We have studied the intrinsic fluorescence of the 12 tryptophan residues of electron-transfer flavoprotein:ubiquinone oxidoreductase (ETF:QO). The fluorescence emission spectrum (lambda ex 295 nm) showed that the fluorescence is due to the tryptophan residues and that the contribution of the 22 tyrosine residues is minor. The emission maximum (lambda m 334 nm) and the bandwidth (delta lambda 1/2 56 nm) suggest that the tryptophans lie in hydrophobic environments in the oxidized protein. Further, these tryptophans are inaccessible to a range of ionic and nonionic collisional quenching agents, indicating that they are buried in the protein. Enzymatic or chemical reduction of ETF:QO results in a 5% increase in fluorescence with no change of lambda m or delta lambda 1/2. This change is reversible upon reoxidation and is likely to reflect a conformational change in the protein. The ubiquinone analogue Q0(CH2)10Br, a pseudosubstrate of ETF:QO (Km = 2.6 microM; kcat = 210 s-1), specifically quenches the fluorescence of one tryptophan residue (Kd = 1.6-3.2 microM) in equilibrium fluorescence titrations. The ubiquinone homologue UQ-2 (Km = 2 microM; kcat = 162 s-1) and the analogue Q0(CH2)10OH (Km = 2 microM; kcat = 132 s-1) do not quench tryptophan fluorescence; thus the brominated analogue acts as a static heavy atom quencher. We also describe a rapid purification for ETF:QO based on extraction of liver submitochondrial particles with Triton X-100 and three chromatographic steps, which results in yields 3 times higher than previously published methods.

Skeletal muscle mitochondrial beta-oxidation. A study of the products of oxidation of [U-14C]hexadecanoate by h.p.l.c. using continuous on-line radiochemical detection.

Well-coupled mitochondrial fractions were prepared from rat skeletal muscle without the use of proteolytic enzymes. The products of [U-14C]hexadecanoate oxidation by rat skeletal muscle mitochondrial fractions were analysed by h.p.l.c. with on-line radiochemical detection. In the presence of 1 mM-carnitine, 70% of the products is acetylcarnitine. In agreement with Veerkamp et al. [Veerkamp, van Moerkerk, Glatz, Zuurveld, Jacobs & Wagenmakers (1986) Biochem. Med. Metab. Biol. 35, 248-259] 14CO2 release is shown to be an unreliable estimate of flux through beta-oxidation in skeletal muscle mitochondrial fractions. The flux through beta-oxidation is recorded unambiguously polarographically in the presence of 1 mM-carnitine and the absence of citrate cycle intermediates.

Defects of fatty acid oxidation in skeletal muscle.

Fatty acids are important substrates for resting and exercising skeletal muscle. Defects of fatty acid oxidation result in muscle pain and weakness, and there is often lipid accumulation in muscle fibres. Only a few of the several possible enzyme defects have been found and some of these have responded to therapy. Some techniques for the investigation of fatty acid oxidation are outlined.

A two-state analysis of co-operative oxygen binding in the three human embryonic haemoglobins.

The binding of oxygen to the three human embryonic haemoglobins, at pH 7.4, has been shown to occur as a co-operative process. Analysis of oxygen-binding curves obtained in the absence of organic phosphate allosteric effectors shows that the process can be described quite accurately by the two-state model of allosteric action. In the presence of organic phosphates, the binding affinity for oxygen to the T-state of the alpha 2 epsilon 2 and zeta 2 epsilon 2 haemoglobins is significantly lowered. The values of the best-fit two-state parameters determined for each of the embryonic haemoglobins together with the temperature-dependence of the overall equilibrium binding process are discussed in terms of oxygen transfer from the maternal blood supply. Fast-reaction studies have been used to determine the rate constants of the oxygen association and dissociation processes occurring in the R-state and the rate of the allosteric R > T conformational transition. Analysis of these data suggests a likely reason for the high affinity and low co-operativity of the embryonic proteins and identifies the origins of the inability of equilibrium measurements to identify chain non-equivalence in the R-state.

Cytochrome bo from Escherichia coli: binding of azide to CuB.

Azide binds to fast cytochrome bo with a stoichiometry of 1:1, the dissociation constant for this reaction being approximately 2 x 10(-5) M. The changes induced in the electronic absorption are very slight and are consistent with heme o remaining hexacoordinate high-spin, an observation confirmed by room temperature MCD spectroscopy in the region 350-2000 nm. X-band EPR spectroscopy of the azide-bound form shows heme o remains coupled to CuB, but that the integer spin signal (g = 3.7) that we have previously reported to be associated with the binuclear center of fast cytochrome bo [Watmough et al. (1993) FEBS Lett. 319, 151-154], is shifted to higher field. The kinetics of azide binding are an order of magnitude faster than those observed for the binding of cyanide. Unlike cyanide, the observed rate constants do not saturate in the range 0.05-25 mM. The value of Kon shows a marked dependence on pH, indicating that the active species is hydrazoic acid. It is argued that these data are consistent with the binding of azide ion as a terminal ligand to CuB yielding a binuclear center in the form FeIII-OH2:: CuBII-N3. The binding of azide in heme-copper oxidases may cause displacement of another nitrogenous ligand from CuB which might explain the absence of electron density associated with histidine-325 in the structure of the Paracoccus denitrificans CCO [Iwata et al. (1995) Nature 376, 660-669]. Formate appears to act as a bidentate ligand to the binuclear center-, blocking not only the binding of azide to CuB but also the binding of cyanide to heme o.