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PubMed1995

8-Azido-ATP modification of cytochrome c: retardation of its electron-transfer activity to cytochrome c oxidase.

Horse heart cytochrome c has been modified by 8-azido-ATP and the electron-transfer activity of the modified cytochrome c's to bovine heart cytochrome c oxidase (CcO) under physiological ionic strengths has been studied by the laser flash photolysis technique with 5-deazariboflavin and EDTA as the electron donor. The intermolecular electron transfer between the redox protein partners was shown to be extremely slow. The 8-azido-ATP-modified system exhibited less than 5% of the intracomplex electron-transfer rate observed between native cytochrome c and CcO under otherwise identical conditions. The binding affinity of the modified cytochrome c was greatly reduced (3 orders of magnitude) at low ionic strengths; however, it was only slightly reduced (by a factor of 2) relative to the native protein at physiological ionic strengths. Thus, the binding affinity of the ATP-cytochrome c adducts is relatively insensitive to the ionic strength compared to the native enzyme, suggesting that a different docking conformation is assumed by the ATP-cytochrome c adducts in their interaction with the oxidase. Since the redox potential of the modified cytochrome c is close to the value of its native form, we conclude that there has been a change in the docking of the cytochrome c to CcO and the electronic coupling between heme c and CuA upon 8-azido-ATP modification.

Adenosine Triphosphate↗
PubMed1992

Interaction of cytochrome c with cytochrome c oxidase studied by monoclonal antibodies and a protein modifying reagent.

C/57 black mice were immunized with beef heart cytochrome c oxidase, generating 48 hybrid cell lines that secrete antibodies against the different subunits of the enzyme. Immunoblot analysis showed reactions with 7 of the 13 subunits. Among the monoclonal antibodies produced, only those to subunit II gave significant inhibition; these inhibited the enzyme activity completely and prevented cytochrome c binding to the enzyme. Epitope mapping studies indicate that a peptide including residues 200-227 reacts with the antibody, suggesting that the C-terminus of the protein is essential for the binding of this antibody. The carboxyl modifying reagent 1-ethyl-3-[3-(trimethylammonio)propyl]carbodiimide (ETC) was chosen to investigate further the relationship between antibody and cytochrome c binding domains. ETC caused 50% inhibition of the enzyme activity with a first-order time during the first 20 min; a slower reaction over 3 h resulted in 90% inhibition. Cytochrome c binding to the oxidase was inhibited to a similar extent as cytochrome c oxidation, and protection against both effects was afforded by the presence of cytochrome c during ETC modification. Anion-exchange of FPLC of the modified forms of cytochrome oxidase revealed extensive inhomogeneity, indicating random derivatization of a number of different carboxyls even during the first-order reaction, and precluding identification of carboxyl residues related to a specific phase of the reaction. Cytochrome c and the subunit II-specific antibody protected against radioactive labeling of subunit II by ETC in the presence of [14C]glycine ethyl ester, demonstrating that the antibody and cytochrome c occupy significant and overlapping areas on the subunit II surface.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1994

Kinetic mechanism for the model reaction of NADPH-cytochrome P450 oxidoreductase with cytochrome c.

The kinetic mechanism of NADPH-cytochrome P450 oxidoreductase (P450R) has been determined for the model reaction with cytochrome c3+. Although initial velocity studies show parallel patterns, consistent with a classical (one-site) ping-pong mechanism that precludes the formation of a ternary NADPH-P450R-cytochrome c3+ complex, product and dead-end inhibition results suggest a nonclassical (two-site) ping-pong mechanism [Northrop, D. B. (1969) J. Biol. Chem. 244, 5808-5819]. This mechanism is a hybrid of the random sequential (ternary complex) and ping-pong mechanisms, since ternary complexes can form as well as intermediate, modified forms of the enzyme that can be present in the absence of any bound substrate. The complete rate equation is derived for this mechanism, and values for Vmax, (V/K)NADPH, (V/K)cytc, and the corresponding Michaelis constants are presented in terms of microscopic rate constants along with the expected product inhibition patterns (Appendix). Inhibition by NADP+ is competitive versus NADPH and uncompetitive versus cytochrome c3+, while inhibition by cytochrome c2+ is competitive versus cytochrome c3+ and noncompetitive versus NADPH. These inhibition patterns are consistent with the proposed two-site mechanism. This mechanism would give the same initial velocity patterns as the classical one-site ping-pong mechanism, but it allows for the formation of a ternary complex, with NADPH and cytochrome c3+ reacting independently at two separate sites on P450R. The D(V/K)NADPH isotope effect is not affected by cytochrome c3+ concentration, consistent with our assumption (in deriving the rate equation) that binding at the two sites is independent. At the high ionic strength used in this study (850 mM), the mechanism is two-site ping-pong, with the electron acceptor site itself reacting with cytochrome c3+ in a tetra uni ping-pong manner.

Animals↗
PubMed1990

Conformational changes in cytochrome c and cytochrome oxidase upon complex formation: a resonance Raman study.

The fully oxidized complex of cytochrome c and cytochrome oxidase formed at low ionic strength was studied by resonance Raman spectroscopy. The spectra of the complex and of the individual components were compared over a wide frequency range using Soret band excitation. In both partners of the complex, structural changes occur in the heme groups and in their immediate protein environment. The spectra of the complex in the 1600-1700 cm-1 frequency range were dominated by bands from the cytochrome oxidase component, whereas those in the 300-500 cm-1 range were dominated by bands from the cytochrome c component, hence allowing separation of the contributions from the two individual species. For cytochrome c, spectral changes were observed which correspond to the induction of the conformational state I and the six-coordinated low-spin configuration of state II on binding to cytochrome oxidase. While in state I the structure of cytochrome c is essentially the same as in solution, state II is characterized by a structural rearrangement of the heme pocket, leading to a weakening of the axial iron-methionine bond and an opening of the heme crevice which is situated in the center of the binding domain for cytochrome oxidase. The relative contributions of the two cytochrome c states were estimated to be approximately in the ratio 1:1 in the complex.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1977

Effect of specific trifluoroacetylation of individual cytochrome c lysines on the reaction with cytochrome oxidase.

We have prepared three different cytochrome c derivatives, each containing a single specifically trifluoroacetylated lysine at residues 13, 55, and 99, respectively. The only modification that affected cytochrome c oxidase (EC 1.9.3.1) activity was that of lysine-13 at the top of the heme crevice. Trifluoroacetylation of lysine-13 increased the apparent Michaelis constant fivefold compared to that of native cytochrome c, but did not affect the maximum velocity. Trifluoroacetylation of lysine-55 at the left side of the cytochrome c molecule did not affect cytochrome oxidase activity in any way, nor did trifluoroacetylation of lysine-99 at the rear of the cytochrome c molecule. This indicates that the cytochrome oxidase binding site on cytochrome c involved only the front of the cytochrome c molecule and those lysines immediately surrounding the heme crevice.

Animals↗
PubMed1975

Conformational and functional studies of chemically modified cytochrome c: nitrated and iodinated cytochromes c.

The purification of iodinated (E. B. McGowan and E. Stellwagen (1970), Biochemistry 9, 3074) and of nitrated (M. Sokolovsky et al. (1970), Biochemistry 9, 5113) cytochromes c resulted in the recovery from the former preparation of diiododityrosyl-cytochrome c (DIDT-) with modification of Tyr-67 and Tyr-74, and, from the latter, a mononitromonotyrosyl-cytochrome c (MNMT-), with modification of Tyr-67, and mononitrodityrosyl-cytochrome c (MNDT-), with the added modification of Tyr-48. The three purified preparations were conformationally characterized using pH-spectroscopy, circular dichroism, thermal denaturation, reducibility with ascorbate, autoxidation with molecular oxygen, and binding with CO. These results are related to the two aspects of biological function, reducibility, measured by NADH-cytochrome c reductase, and oxidizability, with cytochrome c oxidase, as well as to structure-function relationships in the protein. MNMT-cytochrome c was found to be, structurally and conformationally, a single isomer, reducible with ascorbate, with a small, but definite affinity for both oxidation with molecular oxygen and binding of CO. Conformationally, in both valence states of the metal atom, it represents a molecular form with native-like conformation with small but definite perturbations in the immediate vicinity of the heme group, reflected by the destabilization of the Met-80-S-Fe linkage. MNMT-ferricytochrome c exhibits a pK of 6.2 for the transformation of the low-spin, native-like spectral form II containing the 695-nm band to form lacking lacking the 695-nm band. The isomerization at pK = 6.2, when analyzed in terms of the isomerization of the native protein with a pK of 9.2 and the nature of the group involved, indicates that Tyr-67 is not involved in the isomerization of the modified preparation, and possibly not in the native protein as well. In terms of biological function, the partial derangement of redecibility (24%) and the unaltered oxidizability point to the functional significance of Tyr-67, and provide another example of selectivity between the two aspects of physiological functional function, in agreement with the two-function, two-path operational model of the protein. The MNDT- and DIDT-ferricytochromes c exhibited physicochemical properties indicative of gross derangement of both the conformation of the protein as well as of the coordination configuration of the metal atom. The complete inability to accept an electron from NADH-cytochrome c reductase in both cases, and the retention of 50% of the oxidizability property of DIDT-cytochrome c, were interpreted to be the result of conformational derangement, rather than the added modification of Tyr-48 or of Tyr-74.

Amino Acids↗
PubMed2003

Design of a ruthenium-labeled cytochrome c derivative to study electron transfer with the cytochrome bc1 complex.

A new ruthenium-cytochrome c derivative was designed to study electron transfer from cytochrome bc1 to cytochrome c (Cc). The single sulfhydryl on yeast H39C;C102T iso-1-Cc was labeled with Ru(2,2'-bipyrazine)2(4-bromomethyl-4'-methyl-2,2'-bipyridine) to form Ru(z)-39-Cc. The Ru(z)-39-Cc derivative has the same steady-state activity with yeast cytochrome bc1 as wild-type yeast iso-1-Cc, indicating that the ruthenium complex does not interfere in the binding interaction. Laser excitation of reduced Ru(z)-39-Cc results in electron transfer from heme c to the excited state of ruthenium with a rate constant of 1.5 x 10(6) x s(-1). The resulting Ru(I) is rapidly oxidized by atmospheric oxygen in the buffer. The yield of photooxidized heme c is 20% in a single flash. Flash photolysis of a 1:1 complex between reduced yeast cytochrome bc1 and Ru(z)-39-Cc at low ionic strength leads to rapid photooxidation of heme c, followed by intracomplex electron transfer from cytochrome c1 to heme c with a rate constant of 1.4 x 10(4) x s(-1). As the ionic strength is raised above 100 mM, the intracomplex phase disappears, and a new phase appears due to the bimolecular reaction between solution Ru-39-Cc and cytochrome bc1. The interaction of yeast Ru-39-Cc with yeast cytochrome bc1 is stronger than that of horse Ru-39-Cc with bovine cytochrome bc1, suggesting that nonpolar interactions are stronger in the yeast system.

Crystallography, X-Ray↗
PubMed1979

Structure of cytochrome a3-Cua3 couple in cytochrome c oxidase as revealed by nitric oxide binding studies.

The addition of NO to oxidized cytochrome c oxidase (ferrocytochrome c:oxygen oxidoreductase, EC 1.9.3.1) causes the appearance of a high-spin heme electron paramagnetic resonance (EPR) signal due to cytochrome a3. This suggests that NO coordinates to Cu+2a3 and breaks the antiferromagnetic couple by forming a cytochrome a+33-Cu+2a3-NO complex. The intensity of the high-spin cytochrome a3 signal depends on the method of preparation of the enzyme and maximally accounts for 58% of one heme. The effect of N-3 on the cytochrome a+33-Cu+2a3-NO complex is to reduce cytochrome a3 to the ferrous state, and this is followed by formation of a new complex that exhibits EPR signals characteristic of a triplet species. On the basis of optical and EPR results, a NO bridge between cytochrome a+23 and Cu+2a3 is proposed--i.e., cytochrome a+23-NO-Cu+2a3. The half-field transition observed at g = 4.34 in the EPR spectrum of this triplet species exhibits resolved copper hyperfine splittings with [A+2] = 0.020 cm-1, indicating that the Cu+2a3 in the cytochrome a+23-NO-Cu+2a3 complex is similar to a type 2 copper site.

Animals↗
PubMed1999

Ubiquinol:cytochrome c oxidoreductase. Effects of inhibitors on reverse electron transfer from the iron-sulfur protein to cytochrome b.

The effects of inhibitors on the reduction of the bis-heme cytochrome b of ubiquinol: cytochrome c oxidoreductase (complex III, bc1 complex) has been studied in bovine heart submitochondrial particles (SMP) when cytochrome b was reduced by NADH and succinate via the ubiquinone (Q) pool or by ascorbate plus N,N,N', N'-tetramethyl-p-phenylenediamine via cytochrome c1 and the iron-sulfur protein of complex III (ISP). The inhibitors used were antimycin (an N-side inhibitor), beta-methoxyacrylate derivatives, stigmatellin (P-side inhibitors), and ethoxyformic anhydride, which modifies essential histidyl residues in ISP. In agreement with our previous findings, the following results were obtained: (i) When ISP/cytochrome c1 were prereduced or SMP were treated with a P-side inhibitor, the high potential heme bH was fully and rapidly reduced by NADH or succinate, whereas the low potential heme bL was only partially reduced. (ii) Reverse electron transfer from ISP/c1 to cytochrome b was inhibited more by antimycin than by the P-side inhibitors. This reverse electron transfer was unaffected when, instead of normal SMP, Q-extracted SMP containing 200-fold less Q (0. 06 mol Q/mol cytochrome b or c1) were used. (iii) The cytochrome b reduced by reverse electron transfer through the leak of a P-side inhibitor was rapidly oxidized upon subsequent addition of antimycin. This antimycin-induced reoxidation did not happen when Q-extracted SMP were used. The implications of these results on the path of electrons in complex III, on oxidant-induced extra cytochrome b reduction, and on the inhibition of forward electron transfer to cytochrome b by a P-side plus an N-side inhibitor have been discussed.

Animals↗
PubMed1999

Cytochrome c550 is an essential component of the quinoprotein ethanol oxidation system in Pseudomonas aeruginosa: cloning and sequencing of the genes encoding cytochrome c550 and an adjacent acetaldehyde dehydrogenase.

Pseudomonas aeruginosa ATCC 17933 grown aerobically on ethanol produces a soluble cytochrome c550 together with a quinoprotein ethanol dehydrogenase. A 3.2 kb genomic DNA fragment containing the gene encoding cytochrome c550 was cloned and sequenced. Two other complete and two truncated ORFs were also identified. A truncated ORF encoding the quinoprotein ethanol dehydrogenase (exaA) was found upstream of the cytochrome c550 gene (exaB) and in reverse orientation. An ORF encoding a NAD(+)-dependent acetaldehyde dehydrogenase (exaC) was located downstream of the cytochrome c550 gene and in the same orientation. Another ORF showed similarity to the pqqA gene and a truncated ORF similarity to the pqqB gene, both involved in the biosynthesis of the prosthetic group PQQ. The organization of these genes was found to be different from the well-studied methanol oxidation system in methylotrophic bacteria. The deduced amino acid sequence of cytochrome c550 from P. aeruginosa showed some similarity to cytochrome c6 of the alga Chlamydomonas reinhardtii and the haem domain of quinohaemoprotein alcohol dehydrogenases of acetic acid bacteria, but no similarity to the soluble cytochrome cL of the quinoprotein methanol oxidation system of methylotrophs could be detected. A mutant of P. aeruginosa with an interrupted cytochrome c550 gene was unable to grow on ethanol, which proves that cytochrome c550 is an essential component of the ethanol oxidation system in this organism.

Aldehyde Oxidoreductases↗
PubMed2000

Integrity of thermus thermophilus cytochrome c552 synthesized by Escherichia coli cells expressing the host-specific cytochrome c maturation genes, ccmABCDEFGH: biochemical, spectral, and structural characterization of the recombinant protein.

We describe the design of Escherichia coli cells that synthesize a structurally perfect, recombinant cytochrome c from the Thermus thermophilus cytochrome c552 gene. Key features are (1) construction of a plasmid-borne, chimeric cycA gene encoding an Escherichia coli-compatible, N-terminal signal sequence (MetLysIleSerIleTyrAlaThrLeu AlaAlaLeuSerLeuAlaLeuProAlaGlyAla) followed by the amino acid sequence of mature Thermus cytochrome c552; and (2) coexpression of the chimeric cycA gene with plasmid-borne, host-specific cytochrome c maturation genes (ccmABCDEFGH). Approximately 1 mg of purified protein is obtained from 1 L of culture medium. The recombinant protein, cytochrome rsC552, and native cytochrome c552 have identical redox potentials and are equally active as electron transfer substrates toward cytochrome ba3, a Thermus heme-copper oxidase. Native and recombinant cytochromes c were compared and found to be identical using circular dichroism, optical absorption, resonance Raman, and 500 MHz 1H-NMR spectroscopies. The 1.7 A resolution X-ray crystallographic structure of the recombinant protein was determined and is indistinguishable from that reported for the native protein (Than, ME, Hof P, Huber R, Bourenkov GP, Bartunik HD, Buse G, Soulimane T, 1997, J Mol Biol 271:629-644). This approach may be generally useful for expression of alien cytochrome c genes in E. coli.

Amino Acid Sequence↗
PubMed1977

Metal-free and metal-substituted cytochromes c. Use in characterization of the cytochrome c binding site.

The luminescent properties of metal-free, tin(IV) and zinc(II) cytochromes c have been used to characterize the interaction of cytochrome c with mitochondria and cytochrome oxidase. Diminution in the fluorescence yields of tin and zinc cytochrome c occur when these derivates bind to cytochrome oxidase or mitochondria. Based upon spectral overlap and quantum yield, the distance between the porphyrin rings of cytochrome a and cytochrome c is estimated according to Forster theory to be in the neighborhood of 3.5 nm. Measurements of the polarized emission of metal-free 'porphyrin' cytochrome c when bound to oriented layers of cytochrome c oxidase indicate that the porphyrin is bound obliquely to the plane of the oxidase layers with an angle of about 70 degrees C from heme plane to membrane plane. It is proposed that these data have significance for elucidation of electron transfer mechanisms.

Animals↗
PubMed1989

Circular dichroic spectroscopy of membrane haemoproteins. The molecular determinants of the dichroic properties of the b cytochromes in various ubiquinol:cytochrome c reductases.

The circular dichroism (CD) of dihaem cytochrome b from mitochondrial and bacterial ubiquinol:cytochrome-c reductase (bc1 complex) has been characterized. The dichroic properties of the yeast purified cyt b are very similar to those of the native cyt b within the mitochondrial bc1 complex. The CD spectra in the Soret region of the native cytochrome b present in all species studied show an intense bisignate Cotton effect having a zero-crossing wavelength close to the absorbance maximum. In preparations partially or completely depleted of the low-potential b haem (b1) the CD spectra exhibit a single positive Cotton effect resembling the corresponding absorption spectrum. This is particularly evident in the purified cytochrome b-562 from Rhodobacter sphaeroides R26, which contains only the high-potential b haem (bh). These spectral features together with the reconstitution of the cytochrome b1 haem have been used to resolve the CD contribution of each haem to the CD spectra of cytochrome b. The mechanisms which might be responsible for the optical activity have been examined. It appears that the CD spectra of cytochrome b derive from both the mutual interaction of its two haems (giving rise to exciton coupling) and to the interaction of each haem with nearby aromatic residues, other than the pairs of histidines which coordinate the iron. The dipole coupling between haem and aromatic residues appears to be more important than exciton coupling in the CD spectra of oxidized b cytochromes and correlations have been made between the CD features and the proposed structure of cytochrome b.

Animals↗
PubMed2003

The dithiol:disulfide oxidoreductases DsbA and DsbB of Rhodobacter capsulatus are not directly involved in cytochrome c biogenesis, but their inactivation restores the cytochrome c biogenesis defect of CcdA-null mutants.

The cytoplasmic membrane protein CcdA and its homologues in other species, such as DsbD of Escherichia coli, are thought to supply the reducing equivalents required for the biogenesis of c-type cytochromes that occurs in the periplasm of gram-negative bacteria. CcdA-null mutants of the facultative phototroph Rhodobacter capsulatus are unable to grow under photosynthetic conditions (Ps(-)) and do not produce any active cytochrome c oxidase (Nadi(-)) due to a pleiotropic cytochrome c deficiency. However, under photosynthetic or respiratory growth conditions, these mutants revert frequently to yield Ps(+) Nadi(+) colonies that produce c-type cytochromes despite the absence of CcdA. Complementation of a CcdA-null mutant for the Ps(+) growth phenotype was attempted by using a genomic library constructed with chromosomal DNA from a revertant. No complementation was observed, but plasmids that rescued a CcdA-null mutant for photosynthetic growth by homologous recombination were recovered. Analysis of one such plasmid revealed that the rescue ability was mediated by open reading frame 3149, encoding the dithiol:disulfide oxidoreductase DsbA. DNA sequence data revealed that the dsbA allele on the rescuing plasmid contained a frameshift mutation expected to produce a truncated, nonfunctional DsbA. Indeed, a dsbA ccdA double mutant was shown to be Ps(+) Nadi(+), establishing that in R. capsulatus the inactivation of dsbA suppresses the c-type cytochrome deficiency due to the absence of ccdA. Next, the ability of the wild-type dsbA allele to suppress the Ps(+) growth phenotype of the dsbA ccdA double mutant was exploited to isolate dsbA-independent ccdA revertants. Sequence analysis revealed that these revertants carried mutations in dsbB and that their Ps(+) phenotypes could be suppressed by the wild-type allele of dsbB. As with dsbA, a dsbB ccdA double mutant was also Ps(+) Nadi(+) and produced c-type cytochromes. Therefore, the absence of either DsbA or DsbB restores c-type cytochrome biogenesis in the absence of CcdA. Finally, it was also found that the DsbA-null and DsbB-null single mutants of R. capsulatus are Ps(+) and produce c-type cytochromes, unlike their E. coli counterparts, but are impaired for growth under respiratory conditions. This finding demonstrates that in R. capsulatus the dithiol:disulfide oxidoreductases DsbA and DsbB are not essential for cytochrome c biogenesis even though they are important for respiration under certain conditions.

Amino Acid Sequence↗
PubMed1975

The intratesticular localization of cytochrome P-450 and cytochrome P-450-dependent enzymes in the rat testis.

Following separation of the seminiferous tubules from the interstitial cells in the rat testis, the amount of cytochrome P-450 and the activities of the cytochrome P-450-dependent enzymes, the 17alpha-hydroxylase and the C17-C20 lyase, were measured in the microsomes of the separated fractions. The amount of cytochrome P-450-dependent enzymes recovered in the microsomal fraction of the interstitial cells ranged from 71 to 86% of the whole testis. However, in some experiments lower recoveries of the activities of the enzymes were attributed to the breakdown of cytochrome P-450 to cytochrome P-420. In all cases, less than 10% of the testicular cytochrome P-450 and the cytochrome P-450-dependent steroidogenic enzymes were found in the tubular microsomes. Moreover, the specific activities of the 17 alpha-hydroxylase and the C17-C20 lyase were found to be 10 to 30 times higher in the interstitial tissue than in the seminiferous tubules of the rat testis. From these results, we have concluded that cytochrome P-450 and the activities of the cytochrome P-450-dependent enzymes in the rat testis are predominantly, if not sole, located in the interstitial cells.

Animals↗
PubMed1977

Multiple low spin forms of the cytochrome c ferrihemochrome. EPR spectra of various eukaryotic and prokaryotic cytochromes c.

1. Despite the same methionine-sulfur:heme-iron:imidazole-nitrogen hemochrome structure observed by x-ray crystallography in four of the seven c-type eukaryotic and prokaryotic cytochromes examined, and the occurrence of the characteristic 695 nm absorption band correlated with the presence of a methionine-sulfur:heme-iron axial ligand in all seven proteins, they fall into two distinct classes on the basis of their EPR and optical spectra. The horse, tuna, and bakers' yeast iso-1 cytochromes c have a predominant neutral pH EPR form with g1=3.06, g2=2.26, and g3=1.25, while the bakers' yeast iso-2 and Euglena cytochromes c, the Rhodospirillum rubrum cytochrome c2, and the Paracoccus denitrificans cytochrome c550 all have a predominant neutral pH EPR form with g1=3.2, g2=2.05, and g3=1.39. The ferricytochromes with g1=3.06 have a B-Q splitting that is approximately 150 cm-1 larger than the ferricytochromes with g1=3.2. 2. Each of the cytochromes displays up to four low spin EPR forms that are in pH-dependent equilibrium and can all be observed at near neutral pH. As the pH is raised the predominant neutral pH form is converted into two forms with g1=3.4 and g1=3.6, identified by comparsion with model compounds and other heme proteins as epsilon-amino:heme-iron:imidazole and bis-epsilon-amino:heme-iron ferrihemochromes, respectively. 3. The pK for the conversion of the predominant neutral pH EPR form into the alkaline pH forms is the same as the pK for the disappearance of the 695 nm absorption band for the cytochromes, even though these pK values range over 2 pH units. This confirms that the g1=3.06 and g1=3.2 forms contain the methionine-sulfur:heme-iron axial ligand while the g1=3.4 and the g1=3.6 forms do not. 4. At extremes of pH, the horse and bakers' yeast iso-1 proteins display several high and low spin forms that are identified, showing that a variety of protein-derived ligands will coordinate to the heme iron including methionine and cysteine sulfur, histidine imidazole, and lysine epsilon-amine. 5. The spectrum of horse cytochrome c with added azide, cyanide, hydroxide, or imidazole as axial ligands has also been examined. 6. From a comparison of the EPR and optical spectral characteristics of these groups of cytochromes with model compounds, it is suggested that the difference between them is due to a change in the hydrogen bonding or perhaps even in the protonation of N-1 of the heme iron-bound histidine imidazole.

Animals↗
PubMed1987

Altered regulation of cytochrome P-450 enzymes in choline-deficient cirrhotic male rat liver: impaired regulation and activity of the male-specific androst-4-ene-3,17-dione 16 alpha-hydroxylase, cytochrome P-450UT-A, in hepatic cirrhosis.

Total microsomal cytochrome P-450 levels were decreased, to about 50% of control, in liver of male rats made cirrhotic by the prolonged intake of a choline-deficient diet. We have suggested previously that this decrease in cytochrome P-450 levels is not a generalized one, but is selective for certain forms of the enzyme. In the present study, levels of six cytochrome P-450 forms including the sex-specific cytochrome P-450 forms, P-450UT-A, P-450PCN-E, and P-450UT-l, were quantitated immunochemically in hepatic microsomes prepared from control and cirrhotic male rats and were related to changes in the regioselectivity of cytochrome P-450-mediated androst-4-ene-3,17-dione hydroxylation in these fractions. The principal finding of this study was that the male-specific androst-4-ene-3,17-dione 16 alpha-hydroxylase was decreased in cirrhotic microsomes to about 20% of control. The content of P-450UT-A decreased concurrently from about 0.40 to less than 0.01 nmol/mg of microsomal protein. Other pathways of androst-4-ene-3,17-dione hydroxylation were also affected, but to different extents than the 16 alpha-hydroxylase. 6 beta-Hydroxylation decreased in cirrhotic microsomes to about 45% of control, despite a marked decrease in P-450PCN-E from 0.27 to less than 0.002 nmol/mg of microsomal protein. The rate of androst-4-ene-3,17-dione 7 alpha-hydroxylation underwent a less pronounced reduction in cirrhosis to about two-thirds of control microsomal activity, and levels of the cytochrome P-450 associated with this activity, P-450UT-F, were decreased in proportion with the decrease in total microsomal cytochrome P-450. 16 beta-Hydroxylase activity was unaffected by the cirrhotogenic process. From spectral binding studies it was apparent that androst-4-ene-3,17-dione elicited a high affinity type I interaction in control microsomal fractions (Ks = 4.5 microM), whereas no interaction was apparent in cirrhotic liver microsomes. Levels of three other forms of cytochrome P-450--P-450PB-C (a constitutive form inducible by phenobarbital), P-450ISF-G (a major isosafrole-inducible form), and P-450UT-I (the major female sexually-differentiated isozyme)--were apparently unaltered in cirrhosis. These findings are consistent with the assertion that specific forms of cytochrome P-450 are subject to altered regulation in hepatic cirrhosis.

Androstenedione↗
PubMed1985

Monoclonal antibodies against bovine adrenal cytochrome P-450(11 beta) and cytochrome P-450SCC. Their isolation, characterization and application to immunohistochemical analysis of adrenal cortex.

Seven monoclonal antibodies directed against bovine adrenocortical cytochrome P-45011 beta were isolated. They were found to be immunoglobulins each having distinct affinity to the antigen. Five clones had IgG1 heavy chains, whereas the other two had IgG2b heavy chains. All the clones had kappa light chains. All the monoclonal antibodies recognized a protein among mitochondrial proteins of bovine adrenal cortex, whose mol. wt, 50,000, was the same as that of cytochrome P-45011 beta. Among the monoclonal antibodies isolated, monoclonal antibody 258 recognized a protein of Mr 50,000 in rat adrenal mitochondria, a protein of Mr 47,000 in pig adrenocortical mitochondria, a protein of Mr 50,000 in guinea pig adrenal mitochondria, a protein of Mr 55,000 in rabbit adrenal mitochondria and a protein of Mr 50,000 in human adrenocortical mitochondria. These results suggest that each of these proteins, having a mol. wt around 50,000, recognized by monoclonal antibody 258 is very likely to be the cytochrome P-45011 beta of adrenocortical mitochondria of each animal species. Monoclonal antibody 42718 inhibited by 50% the steroid hydroxylation activity of cytochrome P-45011 beta. In addition to the monoclonal antibodies against cytochrome P-45011 beta, three monoclonal antibodies directed against bovine adrenocortical cytochrome P-450SCC were also isolated and characterized. Immunohistochemical staining of bovine adrenal cortex with the use of these two kinds of monoclonal antibodies revealed that the contents of both cytochrome P-45011 beta and cytochrome P-450SCC were greater in the zona fasciculata and zona reticularis than in the zona glomerulosa. Electron microscopical observation of immunoperoxidase-stained preparations confirmed the presence of these cytochromes in the mitochondria.

Adrenal Cortex↗