A study of the magnetic properties of haem a3 in cytochrome c oxidase by using magnetic-circular-dichroism spectroscopy.

M.c.d. (magnetic-circular-dichroism) spectroscopy was used to study the magnetization properties of the haem centres in cytochrome c oxidase with magnetic fields of between 0 and 5.3 T over the temperature range 1.5--200 K. The oxidized, oxidized cyanide and partially reduced cyanide forms of the enzyme were studied. In the oxidized state only cytochrome a3+ is detectable by m.c.d. spectroscopy, and its magnetization characteristics show it to be a low-spin ferric haem. In the partially reduced cyanide form of the enzyme cytochrome a is in the diamagnetic low-spin ferrous form, whereas cytochrome a3--CN is e.p.r.-detectable and gives an m.c.d.-magnetization curve typical of a low-spin ferric haem. In the oxidized cyanide form of the enzyme both cytochrome a and cytochrome a3--CN are detectable by m.c.d. spectroscopy, although only cytochrome a gives an e.p.r. signal. The magnetization characteristics of haem a3--CN show clearly that its ground state is an electronic doublet and that another state, probably a spin singlet, lies greater than 10 cm-1 above this. These features are well accounted for by an electronic state of spin S = 1 with a predominantly axial distortion, which leaves the doublet, Ms = +/- 1, as the ground state and the component Ms = 0 as the excited state. This state would not give an e.p.r. signal. Such an electronic state could arise either from a ferromagnetic coupling between haem a3+(3)-CN and the cupric ion, Cua3, or form a haem in the Fe(IV) state.     

Cytochrome c'''' isolated from Methylophilus methylotrophus. An example of bis-histidine-co-ordinated Fe3+ haem, with near-perpendicular orientation of the ligands.

Cytochrome c' (Methylophilus methylotrophus) is a soluble protein, Mr 15,000, possessing one haem which is high-spin in the reduced state but switches to a low-spin form on oxidation. Low-temperature electron-paramagnetic-resonance spectroscopy of the oxidized state shows a low-spin signal at gz = 3.65 with a folded line-shape typical of a haem of low rhombicity, and the near-infrared magnetic-circular-dichroism (m.c.d.) spectra reveal an unusually intense (delta epsilon = 400 M-1.cm-1 at 5 T, 4.2 K) charge-transfer band at 1560 nm, establishing that the oxidized haem is co-ordinated by two His residues in a near-perpendicular orientation. This conformation is well established for transmembrane b cytochromes, but this appears to be the first example in a water-soluble cytochrome. The low-temperature m.c.d. spectra of the reduced form of the protein confirms that the haem contains a high-spin Fe2+ ligated by one His residue. The redox-linked spin-state change releases a His group. Since this residue is likely to bind a proton at pH values less than 6.5, this cytochrome may provide a useful model of a molecular mechanism of a redox-linked proton uptake and release process.     

Inhibition by cycloheximide of degradation of cytochrome P-450 in primary cultures of adult rat liver parenchymal cells and in vivo

Degradation of cytochrome P-450 was studied in adult rat liver parenchymal cells in primary monolayer culture. In cells incubated in standard culture medium, the amount of cytochrome P-450 decreased at an accelerated rate relative to either the rate of degradation of total protein in the cells or the turnover of cytochrome P-450 in vivo. This change was succeeded by a spontaneous increase in the activity of haem oxygenase, an enzyme system that converts haem into bilirubin in vitro, measured in extracts from the cultured cells. This finding suggests that the rate of cytochrome P-450 breakdown may be controlled by factor(s) other than the activity of haem oxygenase. The decline in cytochrome P-450 and the subsequent increase in haem oxygenase activity was prevented by incubation of hepatocytes in medium containing an inhibitor of protein synthesis such as cycloheximide, puromycin, actinomycin D, or azaserine. The effect of cycloheximide appeared to be due to decreased breakdown of microsomal (14)C-labelled haem. By contrast, cycloheximide was without effect on the degradation of total protein, measured either in homogenates or in microsomal fractions prepared from the cultured cells. These results suggest that the conditions of cell culture stimulate selective degradation of cytochrome P-450 by a process that is inhibited by cycloheximide and hence may require protein synthesis. The findings in culture were verified in parallel studies of cytochrome P-450 degradation in vivo. After administration of bromobenzene, the degradation of the haem moiety of cytochrome P-450 was accelerated in vivo in a manner resembling that observed in cultured hepatocytes. Administration of cycloheximide to either bromobenzene-treated rats or to untreated rats decreased the degradation of the haem moiety of cytochrome P-450. However, the drug failed to affect degradation of haem not associated with cytochrome P-450, suggesting that cycloheximide is not a general inhibitor of haem oxidation in the liver. These findings confirm that the catabolism of hepatic cytochrome P-450 haem is controlled by similar cycloheximide-sensitive processes in the basal steady state in vivo, as stimulated by bromobenzene in vivo, or in hepatocytes under the conditions of cell culture. We conclude that the rate-limiting step in this process appears to require protein synthesis and precedes cleavage of the haem ring.     

Structural basis for the network of functional cooperativities in cytochrome c(3) from Desulfovibrio gigas: solution structures of the oxidised and reduced states

Cytochrome c(3) is a 14 kDa tetrahaem protein that plays a central role in the bioenergetic metabolism of Desulfovibrio spp. This involves an energy transduction mechanism made possible by a complex network of functional cooperativities between redox and redox/protolytic centres (the redox-Bohr effect), which enables cytochrome c(3) to work as a proton activator. The three-dimensional structures of the oxidised and reduced Desulfovibrio gigas cytochrome c(3) in solution were solved using 2D (1)H-NMR data. The reduced protein structures were calculated using INDYANA, an extended version of DYANA that allows automatic calibration of NOE data. The oxidised protein structure, which includes four paramagnetic centres, was solved using the program PARADYANA, which also includes the structural paramagnetic parameters. In this case, initial structures were used to correct the upper and lower volume restraints for paramagnetic leakage, and angle restraints derived from (13)C Fermi contact shifts of haem moiety substituents were used for the axial histidine ligands. Despite the reduction of the NOE intensities by paramagnetic relaxation, the final family of structures is of similar precision and accuracy to that obtained for the reduced form. Comparison of the two structures shows that, although the global folds of the two families of structures are similar, significant localised differences occur upon change of redox state, some of which could not be detected by comparison with the X-ray structure of the oxidised state: (1) there is a redox-linked concerted rearrangement of Lys80 and Lys90 that results in the stabilisation of haem moieties II and III when both molecules are oxidised or both are reduced, in agreement with the previously measured positive redox cooperativity between these two haem moieties. This cooperativity regulates electron transfer, enabling a two-electron step adapted to the function of cytochromes c(3) as the coupling partner of hydrogenase; and (2) the movement of haem I propionate 13 towards the interior of the protein upon reduction explains the positive redox-Bohr effect, establishing the structural basis for the redox-linked proton activation mechanism necessary for energy conservation, driving ATP synthesis.     

Spectroscopic characterization of a high-potential monohaem cytochrome from Wolinella succinogenes, a nitrate-respiring organism. Redox and spin equilibria studies

When purified, a high-potential c-type monohaem cytochrome from the nitrate-respiring organism, Wollinella succinogenes (VPI 10659), displayed a minimum molecular mass of 8.2 kDa and 0.9 mol iron and 0.95 mol haem groups/mol protein. Visible light spectroscopy suggested the presence of an equilibrium between two ligand arrangements around the haem, i.e. an absorption band at 695 nm characteristic of haem-methionine coordination (low-spin form) coexisting with a high-spin form revealed by a band at 619 nm and a shoulder at 498 nm. The mid-point redox potential measured by visible redox titration of the low-spin form was approximately +100 mV. Binding cyanide (Ka = 5 x 10(5) M-1) resulted in the displacement of the methionyl axial residue, and full conversion to a low-spin, cyanide-bound form. Structural features were studied by 300-MHz 1H-NMR spectroscopy. In the oxidized state, the pH dependence of the haem methyl resonances (pH range 5-10) and the magnetic susceptibility measurements (using an NMR method) were consistent with the visible light spectroscopic data for the presence of a high-spin/low-spin equilibrium with a transition pKa of 7.3. The spin equilibrium was fast on the NMR time scale. The haem methyl resonances presented large downfield chemical shifts. An unusually broad methyl resonance at around 35 ppm (pH = 7.5, 25 degrees C) was extremely temperature-dependent [delta(323 K) - delta(273 K) = 7.2 ppm] and was assigned to the S-CH3 group of the axial methionine. In the ferrous state only a low-spin form is present. The haem meso protons, the methyl group and the methylene protons from the axial methionine were identified in the reduced form. The resonances from the aromatic residues (three tyrosines and one phenylalanine) were also assigned. Detailed monitoring of the NMR-redox pattern of the monohaem cytochrome from the fully reduced up to the fully oxidized state revealed that the rate of the intermolecular electronic exchange process was approximately 6 x 10(6) M-1 s-1 at 303 K and pH = 6.31. A dihaem cytochrome also present in the crude cell extract and purified to a homogeneous state, exhibited a molecular mass of 11 kDa and contained 2.43 mol iron and 1.89 mol haem c moieties/mol cytochrome. The absorption spectrum in the visible region exhibited no band at 695 nm, suggesting that methione is not a ligand for either of the two haems. Recovery of only small amounts of this protein prevented more detailed structural analyzes.     

Redox-linked spin-state changes in the di-haem cytochrome c-551 peroxidase from Pseudomonas aeruginosa.

Magnetic-c.d., e.p.r. and optical-absorption spectra are reported for the half-reduced form of Pseudomonas aeruginosa cytochrome c-551 peroxidase, a di-haem protein, and its fluoride derivative. Comparison of this enzyme species with oxidized peroxidase shows the occurrence of spin-state changes at both haem sites. The high-potential haem changes its state from partially high-spin to low-spin upon reduction. This is linked to a structural alteration at the ferric low-potential haem group, causing it to change from low-spin to high-spin. Low-temperature spectra demonstrate photolysis of an endogenous ligand of the high-potential haem. In addition, an inactive form of enzyme is examined in which the structural change at the ferric low-potential haem does not occur on reduction of the high-potential haem.     

Formation of cytochrome P-450 containing haem or cobalt-protoporphyrin in liver homogenates of rats treated with phenobarbital and allylisopropylacetamide.

The potent porphyrogen allylisopropylacetamide and related compounds decrease hepatic concentrations of cytochrome P-450. This decrease occurs particularly in phenobarbital-induced cytochrome P-450 and is caused by suicidal breakdown of the haem of cytochrome P-450. Quantitative rocket immunoelectrophoresis showed that the protein moiety of the major phenobarbital-inducible form of hepatic cytochrome P-450 was not diminished up to 1 h, but was markedly decreased (to 43% of that of the phenobarbital-treated control) at 20 h after allylisopropylacetamide treatment. In contrast, the concentration of total cytochrome P-450, measured spectrophotometrically, decreased to 30-40% of the control at both 1 and 20 h after allylisopropylacetamide. Cytochrome P-450-dependent demethylations of ethylmorphine and benzphetamine decreased to a similar extent. When liver homogenates from rats treated with allylisopropylacetamide 1 h before being killed were incubated with haem, functional holocytochrome P-450 could be reconstituted from the apoprotein. Incubation with haem increased spectrophotometrically measurable cytochrome P-450 to 69%, ethylmorphine demethylase to 64% and benzphetamine demethylase to 93% of the activities in rats treated with phenobarbital alone. At 20 h after allylisopropylacetamide treatment, however, little or no reconstitution of cytochrome P-450 occurred after incubation with haem. When liver homogenates were incubated with cobalt and protoporphyrin, and microsomal proteins were then subjected to polyacrylamide-gel electrophoresis, cobalt-protoporphyrin was found specifically associated with proteins of Mr 50 000-53 000. When homogenates from rats given allylisopropylacetamide for 1 h or 20 h were compared, it was found that the extent of this association was higher in livers from the rats containing more apocytochrome P-450, suggesting that cobalt-protoporphyrin had associated with the apocytochrome. The data provide insight into the association of haem with the protein moiety of cytochrome P-450 and factors affecting breakdown of this protein.     

Structure and mechanism in the bacterial dihaem cytochrome c peroxidases

The bacterial cytochrome c peroxidases contain an electron-transferring haem c (E) and a peroxidatic haem c (P). Many are isolated in an inactive oxidised state. Reduction of the E haem promotes Ca(2+)-dependent spin state and coordination changes at the P haem rendering it accessible to ligand. Recent crystallographic work on the oxidised and mixed valence enzymes has suggested a mechanism by which an electron entering the E haem remotely triggers this activation of the P haem. Binding of hydrogen peroxide at the activated P haem leads to an intermediate catalytic form containing two oxidising equivalents, one of which is a ferryl oxene. This form of the enzyme is then reduced by two single electron transfers to the E haem delivered by small redox proteins such as cytochromes or cupredoxins. The binding of these small redox proteins is dominated by global electrostatic forces but the interfaces of the electron transfer complexes that are formed are largely hydrophobic and relatively non-specific. These features allow very high electron transfer rates in the steady state.     

Expression, purification and characterization of cytochrome P450 Biol: a novel P450 involved in biotin synthesis in Bacillus subtilis.

The bioI gene has been sub-cloned and over-expressed in Escherichia coli, and the protein purified to homogeneity. The protein is a cytochrome P450, as indicated by its visible spectrum (low-spin haem iron Soret band at 419 nm) and by the characteristic carbon monoxide-induced shift of the Soret band to 448 nm in the reduced form. N-terminal amino acid sequencing and mass spectrometry indicate that the initiator methionine is removed from cytochrome P450 BioI and that the relative molecular mass is 44,732 Da, consistent with that deduced from the gene sequence. SDS-PAGE indicates that the protein is homogeneous after column chromatography on DE-52 and hydroxyapatite, followed by FPLC on a quaternary ammonium ion-exchange column (Q-Sepharose). The purified protein is of mixed spin-state by both electronic spectroscopy and by electron paramagnetic resonance [g values=2.41, 2.24 and 1.97/1.91 (low-spin) and 8.13, 5.92 and 3.47 (high-spin)]. Magnetic circular dichroism and electron paramagnetic resonance studies indicate that P450 BioI has a cysteine-ligated b-type haem iron and the near-IR magnetic circular dichroism band suggests strongly that the sixth ligand bound to the haem iron is water. Resonance Raman spectroscopy identifies vibrational signals typical of cytochrome P450, notably the oxidation state marker v4 at 1,373 cm(-1) (indicating ferric P450 haem) and the splitting of the spin-state marker v3 into two components (1,503 cm(-1) and 1,488 cm(-1)), indicating cytochrome P450 BioI to be a mixture of high- and low-spin forms. Fatty acids were found to bind to cytochrome P450 BioI, with myristic acid (Kd=4.18+/-0.26 microM) and pentadecanoic acid (Kd=3.58+/-0.54 microM) having highest affinity. The fatty acid analogue inhibitor 12-imidazolyldodecanoic acid bound extremely tightly (Kd<1 microM), again indicating strong affinity for fatty acid chains in the P450 active site. Catalytic activity was demonstrated by reconstituting the P450 with either a soluble form of human cytochrome P450 reductase, or a Bacillus subtilis ferredoxin and E. coli ferredoxin reductase. Substrate hydroxylation at the omega-terminal position was demonstrated by turnover of the chromophoric fatty acid para-nitrophenoxydodecanoic acid, and by separation of product from the reaction of P450 BioI with myristic acid.     

Structural evidence for a proton transfer pathway coupled with haem reduction of cytochrome c″ from Methylophilus methylotrophus

The crystal structures of the oxidized and reduced forms of cytochrome c″ from Methylophilus methylotrophus were solved from X-ray synchrotron data to atomic resolution. The overall fold of the molecule in the two redox states is very similar and is comparable to that of the oxygen-binding protein from the purple phototrophic bacterium Rhodobacter sphaeroides. However, significant modifications occur near the haem group, in particular the detachment from axial binding of His95 observed upon reduction as well as the adoption of different conformations of some protonatable residues that form a possible proton path from the haem pocket to the protein surface. These changes are associated with the previously well characterized redox-Bohr behaviour of this protein. Furthermore they provide a model for one of the presently proposed mechanisms of proton translocation in the much more complex protein cytochrome c oxidase.     

The primary and three-dimensional structures of a nine-haem cytochrome c from Desulfovibrio desulfuricans ATCC 27774 reveal a new member of the Hmc family.

BACKGROUND: Haem-containing proteins are directly involved in electron transfer as well as in enzymatic functions. The nine-haem cytochrome c (9Hcc), previously described as having 12 haem groups, was isolated from cells of Desulfovibrio desulfuricans ATCC 27774, grown under both nitrate- and sulphate-respiring conditions. RESULTS: Models for the primary and three-dimensional structures of this cytochrome, containing 292 amino acid residues and nine haem groups, were derived using the multiple wavelength anomalous dispersion phasing method and refined using 1.8 A diffraction data to an R value of 17.0%. The nine haem groups are arranged into two tetrahaem clusters, with Fe-Fe distances and local protein fold similar to tetrahaem cytochromes c3, while the extra haem is located asymmetrically between the two clusters. CONCLUSIONS: This is the first known three-dimensional structure in which multiple copies of a tetrahaem cytochrome c3-like fold are present in the same polypeptide chain. Sequence homology was found between this cytochrome and the C-terminal region (residues 229-514) of the high molecular weight cytochrome c from Desulfovibrio vulgaris Hildenborough (DvH Hmc). A new haem arrangement in domains III and IV of DvH Hmc is proposed. Kinetic experiments showed that 9Hcc can be reduced by the [NiFe] hydrogenase from D. desulfuricans ATCC 27774, but that this reduction is faster in the presence of tetrahaem cytochrome c3. As Hmc has never been found in D. desulfuricans ATCC 27774, we propose that 9Hcc replaces it in this organism and is therefore probably involved in electron transfer across the membrane.     

Effects of mutation of the conserved glutamic acid-286 in subunit I of cytochrome c oxidase from Rhodobacter sphaeroides

We have studied the effects of mutations, E286Q and E286D, of the conserved glutamate in subunit I of cytochrome c oxidase from Rhodobacter sphaeroides with a view to evaluating the role of this residue in redox-linked proton translocation. The mutation E286D did not have any dramatic effects on enzyme properties and retained 50% of wild-type catalytic activity. For E286Q a fraction of the binuclear center was trapped in an unreactive, spectrally distinct form which is most likely due to misfolded protein, but the majority of E286Q reacted normally with formate and cyanide in the oxidized state, and with carbon monoxide and cyanide in the dithionite-reduced form. The mutation also had little effect on the pH-dependent redox properties of haem a in the reactive fraction. However, formation of the P state from oxidized enzyme with hydrogen peroxide or by aerobic incubation with carbon monoxide was inhibited. In particular, only an F-type product was obtained, at less than 25% yield, in the reaction with hydrogen peroxide. The aerobic steady state in the presence of ferrous cytochrome c was characterized by essentially fully reduced haem a and ferric haem a3, suggesting that the mutation hinders electron transfer from haem a to the binuclear center. Under these conditions or after reoxidation, on a seconds time scale, of haem a3 following anaerobiosis, there was no indication of accumulation of significant amounts of P state. We propose that the glutamate is implicated in several steps in the catalytic cycle, O --> R, P --> F, and, possibly, F --> O. The results are discussed in relation to the "glutamate trap" model for proton translocation.     

Artefacts induced on <Emphasis Type="Italic">c</Emphasis>-type haem proteins by electrode surfaces

In this work it is demonstrated that the characterization of c-type haem containing proteins by electrochemical techniques needs to be cautiously performed when using pyrolytic graphite electrodes. An altered form of the cytochromes, which has a redox potential 300 mV lower than that of the native state and displays peroxidatic activity, can be induced by interaction with the pyrolytic graphite electrode. Proper control experiments need to be performed, as altered conformations of the enzymes containing c-type haems can show activity towards the enzyme substrate. The work was focused on the study of the activation mechanism and catalytic activity of cytochrome c peroxidase from Paracoccus pantotrophus. The results could only be interpreted with the assignment of the observed non-turnover and catalytic signals to a non-native conformation state of the electron-transferring haem. The same phenomenon was detected for Met–His monohaem cytochromes (mitochondrial cytochrome c and Desulfovibrio vulgaris cytochrome c-553), as well as for the bis-His multihaem cytochrome c ₃ from Desulfovibrio gigas, showing that this effect is independent of the axial coordination of the c-type haem protein. Thus, the interpretation of electrochemical signals of c-type (multi)haem proteins at pyrolytic graphite electrodes must be carefully performed, to avoid misassignment of the signals and incorrect interpretation of catalytic intermediates.     

Net hepatic and splanchnic metabolism of lactate, pyruvate and propionate in dairy cows in vivo in relation to lactation and nutrient supply.

The significance of the exposed haem edge in cytochrome c was directly probed by chemically modifying the partially exposed haem propionate in the crevice region around residues threonine-78 and threonine-49. Reaction of tuna heart cytochrome c with a water-soluble carbodi-imide at pH 3.7 in the absence of any added nucleophilic base leads to the covalent addition of substituted N-acylureas to the protein at two sites. One site has been shown to be a haem propionate by isotope-tracer and i.r.-spectral analysis of haem purified from the apoprotein. The other site is aspartial acid-62 on the back of the molecule. The modified cytochrome c demonstrates abnormal properties, including auto-oxidizability, a reduction potential of + 105mV, a reversible transition to a high-spin species below pH 5.3, no 695 nm charge-transfer band in the ferric state and abnormal binding to mitochondrial membranes. The derivative does react with cytochrome oxidase in deoxycholate-treated submitochondrial particles or in purified preparations with a specific activity of 43-65% compared with that obtained with native cytochrome c. The results are consistent with the view that an intact haem crevice is essential for normal values for physiochemical characteristics, but the significant residual enzymic activity suggests that the electron-transfer interface and/or the cytochrome oxidase-binding site cannot be localized solely in the region of the exposed haem propionate.     

Incorporation of haemoglobin haem into the rat hepatic haemoproteins tryptophan pyrrolase and cytochrome P-450.

After its administration to intact rats, haemoglobin haem was incorporated into hepatic tryptophan pyrrolase as shown by the marked increase in functional constitution of this enzyme. Incorporation of haemoglobin haem into cytochrome P-450 was demonstrated in intact rats and in the isolated rat liver perfused with haemoglobin-free medium. In both systems, haemoglobin haem restored cytochrome P-450 content and its dependent mixed-function-oxidase activity after substrate-induced destruction of the cytochrome P-450 haem moiety. Further confirmation that haemoglobin haem could be incorporated prosthetically into cytochrome P-450 was achieved by administration of [3H]haemoglobin to rats and subsequent isolation and characterization of radiolabelled substrate-alkylated products of cytochrome P-450 haem. Our findings indicate that, although hepatic uptake of parenteral haemoglobin is slower than that of haem, it appears to serve as an effective haem donor to the intrahepatic 'free' haem pool. Thus parenteral haemoglobin may warrant consideration as a therapeutic alternative to haem in the acute hepatic porphyrias.     

Assembly of cytochrome f into the cytochrome bf complex in isolated pea chloroplasts.

Structural features of cytochrome f necessary for assembly into the cytochrome bf complex were examined in isolated pea chloroplasts following import of (35)S-labelled chimeric precursor proteins, consisting of the presequence of the small subunit of Rubisco fused to the turnip cytochrome f precursor. Assembly was detected by nondenaturing gel electrophoresis of dodecyl maltoside-solubilized thylakoid membranes. A cytochrome f polypeptide unable to bind haem because of mutagenesis of Cys21 and Cys24 to alanine residues was assembled into the complex and had similar stability to the wild-type polypeptide. This indicates that covalent haem binding to cytochrome f is not necessary for assembly of the protein into the cytochrome bf complex. A truncated protein lacking the C-terminal 33 amino acid residues, including the transmembrane span and the stroma-exposed region, was translocated across the thylakoid membrane, had a similar stability to wild-type cytochrome f but was not assembled into the complex. This indicates that the C-terminal region of cytochrome f is important for assembly into the complex. A mutant cytochrome f unable to bind haem and lacking the C-terminal region was also translocated across the thylakoid membrane but was extremely labile, indicating that, in the absence of the C-terminal membrane anchor, haem-less cytochrome f is recognized by a thylakoid proteolytic system.     

Role of haem in the synthesis and assembly of cytochrome P-450

By using 3-amino-1,2,4-triazole, an inhibitor of haem synthesis, and 2-allyl-2-isopropylacetamide, a drug that degrades the haem moiety of cytochrome P-450, the involvement of haem in cytochrome P-450 synthesis and assembly was investigated. Phenobarbital was used to stimulate apo-(cytochrome P-450) synthesis. Degradation of preformed cytochrome P-450 haem does not result in a concomitant release of the apoprotein from the endoplasmic reticulum. The availability of haem for cytochrome P-450 synthesis in the normal animal is not rate-limiting. Prolonged inhibition of haem synthesis in vivo decreases the rate of apo-(cytochrome P-450) synthesis, although this effect is not discernible under conditions of short-term inhibition of haem synthesis. Under the former conditions exogenous haemin is able to counteract the decrease in the rate of apoprotein synthesis. In animals receiving successive injections of phenobarbital plus 3-amino-1,2,4-triazole, compared with those receiving phenobarbital only, the holo-(cytochrome P-450) content measured spectrally shows a greater decrease than could be accounted for by the decrease in the content of the total apoprotein. In addition to less haem being available under these conditions, the free apoprotein appears to have undergone some modification, such that its haem-binding capacity is considerably decreased. This particular effect could be due to a direct interaction of 3-amino-1,2,4-triazole or its metabolites with cytochrome P-450 rather than a consequence of haem deficiency. Apo-(cytochrome P-450) is capable of binding to the endoplasmic reticulum in a form and at a site, which can be reconstituted with haemin to yield the functional protein.     

Synthesis of haem cytochrome c prosthetic group from δ-aminolaevulinate by the cell sap from rat liver

To determine whether the prosthetic group of cytochrome c is synthesized and linked to the apoprotein in the cytosol or in connexion with the endoplasmic reticulum, we have studied the incorporation in vitro of delta-amino[(14)C]laevulinate into porphyrin compounds and cytochrome c by the cell sap from rat liver. The radioactive precursor was incorporated into a trichloroacetic acid-precipitable form partially resistant to extractions by acid solvents, suggesting the existence of a fraction covalently linked to protein. The activity was proportional to the amount of protein incubated, did not increase substantially by supplementation with the microsomal fraction and an energy source, and was very low in the pH5 fraction. Addition of increasing amounts of haemin inhibited the incorporation, as with purified delta-aminolaevulinate dehydratase. [(14)C]Protoporphyrin IX was identified by paper chromatography, together with a shoulder running as protohaem IX. The cell sap in the absence of ribosomes was also able to incorporate radioactivity into purified cytochrome c, and the addition of ribosomes significantly enhanced the activity. The precursors of haem c were synthesized in the soluble system by the known haem-synthetic pathway, as shown by the kinetics of labelling of the coproporphyrin, protoporphyrin and haem fractions, and the activities were concentrated in the precipitate obtained between 40 and 60% saturation with (NH(4))(2)SO(4). The presence of ferrochelatase was indicated by the incorporation of (55)Fe into proto- and haemato-haem identified by paper chromatography. It is concluded that the cell sap from rat liver contains the complete set of enzymes for the synthesis from delta-aminolaevulinate of haem c and its linkage to a small pool of free apoprotein c present in soluble form. This suggests that an ancillary pathway of haem synthesis occurs in the cytosol for at least the formation of the prosthetic group, which is linked post-translationally to that pool of apoprotein c synthesized by free polyribosomes.     

Metal induction of haem oxygenase without concurrent degradation of cytochrome P-450. Protective effects of compound SKF 525A on the haem protein.

The induction of hepatic haem oxygenase (EC 1.14.99.3) by a series of metals, organometals and metalloporphyrins was examined in vivo in the presence of compound SKF 525A, which is known to complex with the prosthetic group of cytochrome P-450. Concurrent administration of SKF 525A and an inducing metal did not affect the extent and time course of haem oxygenase induction. The decrease in cytochrome P-450 content normally associated with metal administration was, however, prevented, indicating that haem oxygenase induction by metals can proceed without the significant labilization of the haem moiety of cytochrome P-450. In addition, the integrity of this haem protein can be maintained by chemical means in the presence of sustained high activities of haem oxygenase.     

Ligand binding to cytochrome c and other related haem proteins and peptides. Part III. Temperature dependence studies

The temperature dependency of ligand binding processes lend support to the proposed mechanisms and the factors affecting ligand binding reported earlier in this series. The free energy contribution from each factor affecting ligand binding was estimated for a number of haem proteins. The structures of the haem proteins used, as conveyed from ligand binding data, are in agreement with the structures of these haem proteins as determined by other methods (e.g. X-ray crystallography, NMR, etc.). Therefore, ligand binding could be used as a facile probe to investigate some of the structural and functional properties of haem proteins. In this respect, it was concluded that the structure of native cytochrome c at pH 10 is similar to the structure of carboxymethyl-Met 80 cytochrome c between pH 7 and 10.