Ingela Jansson

The many roles of cytochrome b5.

Four distinct suggestions have been made to explain the mechanism of the cytochrome b(5)-imposed positive modifier action of the cytochrome P450 monooxygenase reaction. The first mechanism involves a direct input of an electron into the monooxygenase cycle. This is the second of the two electrons necessary for activation of molecular oxygen, and appears to be a rate-limiting step in the monooxygenase reaction. P450 monooxygenases all appear to be uncoupled to varying extents, releasing superoxide and hydrogen peroxide instead of oxidized substrate. A second mechanism suggests that cytochrome b(5) acts as a positive modifier of the monooxygenase by decreasing the extent of uncoupling of the monooxygenase reaction. The implication is that a slow input of the second electron allows uncoupling of a superoxide anion instead of formation of two-electron reduced oxygen. Faster input of the second electron via cytochrome b(5) would result in formation of more of the activated oxygen that reacts with substrate to form product. A third suggestion involves formation of a two-hemoprotein complex between cytochrome b(5) and cytochrome P450 that allows acceptance of two electrons from NADPH-cytochrome P450 reductase. Uncomplexed cytochrome P450 accepts an electron from the reductase, dissociates from it, binds oxygen, and re-associates with the reductase to accept another electron. Complexation with cytochrome b(5) enhances the rate of formation of the active oxygen by obviating the need for two interactions with reductase. The fourth mechanism has cytochrome b(5) serving as an effector without a reduction-oxidation role in the monooxygenation reaction. This effector function may be to enhance the breakdown of the oxygenated hemoprotein to products or to facilitate flow of electrons through the system.

Comparative expression profiling of 40 mouse cytochrome P450 genes in embryonic and adult tissues

This study is the first systematic investigation of the gestational age-dependent and adult tissue-specific expression patterns of each known mouse CYP family (40 genes) using normalized cDNA panels and uniform reverse transcriptase polymerase chain reaction-based assays. Twenty-seven of the P450s were constitutively expressed during development. The number gradually increased through the phases of gastrulation E7 (n=14), neural patterning and somitogenesis E11 (n=17), organogenesis E15 (n=20), and fetal period E17 (n=21). Cyp2s1, Cyp8a1, Cyp20, Cyp21a1, Cyp26a1, Cyp46, and Cyp51 were detected in each of the four stages studied. Members of family CYP1 demonstrated complex, nonoverlapping embryonic patterns of expression, indicating that Cyp1a1 and Cyp1a2 may not compensate for Cyp1b1 deficiency associated with abnormal eye development. Multiple Cyp forms were found to be constitutively expressed in each of the adult tissues studied: liver (n=31), kidney (n=30), testis (n=26), lung (n=24), and heart (n=13). The tissue-specific P450-expression profiles reported in this study provide a reference for more focused analysis of the tissue-specific and developmental functions of the cytochrome P450 monooxygenases.

Studies on three microsomal electron transfer enzyme systems: Specificity of electron flow pathways

Abstract The routes of microsomal electron flow to the three terminal oxidative enzymes, the mixed function oxidase, the fatty acyl CoA desaturase, and the lipid peroxidase have been examined by the use of specific antibodies, by alteration of electron transfer enzyme levels, and with the inhibitor NADP + . From these studies a number of conclusions are drawn: (1) NADH-supported lipid peroxidation utilizes NADH-cytochrome b 5 reductase, but electron flow does not go via cytochrome b 5 . (2) The positive modifier effect of type I substrates on NADPH-driven cytochrome P -450 reduction is seen also with NADH-supported cytochrome P -450 reductase activity. The latter reaction proceeds via cytochrome b 5 while the former does not. (3) Cross-reactivity can occur between NADH-cytochrome b 5 reductase and NADPH-cytochrome c reductase, but at a rate too slow to support most reactions. (4) Cytochrome b 5 appears to exist in two pools; one pool is readily inhibited by antibody and the other pool is either inaccessible to or incompletely inhibited by antibody. The various cytochrome b 5 -dependent reactions show different abilities to use the noninhibited hemoprotein. NADH-cytochrome c reductase activity and NADH-synergism appear to utilize only the former pool and are completely inhibitable by antibody. Other NADH-supported reactions (Δ 9 -desaturation and mixedfunction oxidation) utilize the total cytochrome b 5 population. Fortification studies show that the extra bound cytochrome b 5 is distributed in the same manner as the endogenous cytochrome b 5 .

Effect of two mutations of human CYP1B1, G61E and R469W, on stability and endogenous steroid substrate metabolism.

CYP1B1 is linked to normal eye development by the disease phenotype, primary congenital glaucoma (PCG). CYP1B1 mRNA was expressed in a number of human fetal tissue cDNA libraries, supporting the suggestion of its involvement in tissue development. Highest expression levels were found in thymus and kidney, followed by spleen. A considerably lower level was observed in lung, cardiac and skeletal muscle. No expression was detected in liver or brain. CYP1B1 is able to metabolize steroid hormones. Testosterone was a poor substrate and activity with progesterone was 6-fold higher, but estradiol was the preferred substrate, exhibiting a greater metabolite profile with CYP1B1 than with CYP1A2. Major metabolites were A-ring hydroxylations (75-80%). Others were 15alpha-, 6alpha-, 16alpha- and 6beta-hydroxy metabolites. Two CYP1B1 mutations found in families with the PCG phenotype in which incomplete penetrance is seen were expressed in Escherichia coli. G61E, a hinge region mutation, and R469W, a heme region mutation, were shown to code for holoenzymes. G61E had greatly diminished stability, while the R469W holoenzyme, if anything, was stabilized. Both mutants showed compromised catalytic activity. The extents of isomeric site activity diminution were not proportional, resulting in alterations in the metabolite profiles. The results suggest that if a metabolite of CYP1B1 or elimination of a metabolite by CYP1B1 is necessary for normal embryonic or fetal tissue development, the appearance of these two mutations could result in developmental abnormalities. The altered activities of the mutants and ability of CYP1B1 to respond to external challenge may be the basis for the observed incomplete penetrance.

Spectral analyses of cytochromes P450.

UV/Vis spectroscopy is the major means of identifying intact holocytochrome P450. The carbon monoxide complex of the intact ferrous hemoprotein exhibits a characteristic spectrum between 448 and 452 nm, considerably distinct from the usual Soret absorption peaks of hemoproteins. Methods are described for identification and quantitation of cytochrome P450 (CYP) in membranes, in tissue homogenates, and in purified form, using difference spectroscopy and absolute spectroscopy. CYP are b-type cytochromes, containing protoporphyrin IX as the prosthetic group. Methods are also provided, using alkali and pyridine, for quantitation of the hemoprotein by this prosthetic group. In its oxidized, or ferric state, CYP exists as an equilibrium mixture of high- and low-spin configurations, each with distinctive UV/Vis absorption peaks. Substrate binding causes shifts in the spin equilibrium, and methods are shown for using these shifts for quantitation of substrate binding to CYP.

Epoxidation of styrene by human cyt P450 1A2 by thin film electrolysis and peroxide activation compared to solution reactions.

Films of human cytochrome P450 1A2 (cyt P450 1A2) and polystyrene sulfonate were constructed on carbon cloth electrodes using layer-by-layer alternate absorption and evaluated for electrochemical- and H(2)O(2)-driven enzyme-catalyzed oxidation of styrene to styrene oxide. At -0.6 V vs. saturated calomel reference electrode in an electrochemical cell, epoxidation of styrene was mediated by initial catalytic reduction of dioxygen to H(2)O(2) which activates the enzyme for the catalytic oxidation. Slightly larger turnover rates for cyt P450 1A2 were found for the electrolytic and H(2)O(2) (10 mM) driven reactions compared to conventional enzymatic reactions using cyt P450s, reductases, and electron donors for cytochromes P450 1A2. Cyt P450(cam) gave comparable turnover rates in film electrolysis and solution reactions. Results demonstrate that cyt P450 1A2 catalyzes styrene epoxidation faster than cyt P450(cam), and suggests the usefulness of this thin-film electrolytic method for relative turnover rate studies of cyt P450s.

The many roles of cytochrome b5 in hepatic microsomes

Abstract The purpose of this report is to review the current literature on cytochrome b 5 in hepatic microsomes and to draw conclusions as to its role in microsomal electron transfer pathways. For details concerning the history of cytochrome b 5 the reader is reffered to reviews by C. F. Strittmatter (1) and P. Strittmatter (2). For information on the chemistry of cytochrome b 5 the reader is reffered to the papers by Ozols and Strittmatter (3), Kajihara and Hagihara (4), and Ehrenberg and Bois-Poltoratsky (5). For more recent studies on the isolation and properties of detergent solubilized cytochrome b 5, which contains a hydrophobic peptide enabling reincorporation into membranes, the reader is referred to references 6-12. For simplicity, this minireview is divided into four parts, reflecting areas of study on the role of cytochrome b 5 in the microsomes. One major area is in fatty acid 9 desaturation. Two other areas concern cytochrome b 5 involvement in cytochrome P-450 mediated mixed function oxidations. The fourth section deals with other non-cytochrome P-450 pathways in which cytochrome b 5 is suggested as being a component.

A study of the interaction of a series of substituted barbituric acids with the hepatic microsomal monooxygenase.

1. 1. A series of barbituric acid derivatives have been investigated in an attempt to correlate certain physicochemical parameters, such as lipid solubility and pKa, to their interaction as a substrate with the liver microsomal monooxygenase system. 2. 2. All of the barbiturates gave rise to a type I spectral change when added to suspensions of rat liver microsomes, indicating the formation of a cytochrome P-450-substrate complex. The maximal size of the type I spectral change, however, varied with the different barbiturates and was roughly correlated with their lipid solubility and pKa value. Furthermore, although the allyl-substituted barbiturates produced a type I spectral change at low concentrations, this turned into a modified type II spectral change upon increase in drug concentration. Maximal spectral changes were not additive when different barbiturates in combination were added to the microsomes. 3. 3. Binding affinity for cytochrome P-450, as judged from the spectrally determined dissociation constant (Ks), varied among the different barbiturates studied, and only a rough correlation (correlation coefficient, r = 0.52) was observed between lipid solubility and binding affinity. There was also no good correlation between the rates at which the barbiturates were metabolized by the microsomal monooxygenase system and their lipid solubility (r = 0.42). 4. 4. In contrast, the ability of the barbiturates to act on another membrane-bound enzyme system, the mitochondrial NADH oxidase, revealed a strong correlation (r = 0.92) between lipid solubility and inhibitory efficiency. 5. 5. It is suggested that although lipid solubility is required for a substance to reach the microsomal cytochrome P-450, other properties of the molecule are of importance for determining the affinity with which it will interact with this cytochrome. In the mitochondria, however, lipid solubility alone appears to determine the efficiency with which the various barbiturates exert their inhibitory action on the respiratory chain.

Characterization of the biochemical and structural phenotypes of four CYP1B1 mutations observed in individuals with primary congenital glaucoma.

Objective The objective of this study was to examine the biochemical and physical properties of cytochrome P450 1B1 (CYP1B1) mutants, test our hypothesis that primary congenital glaucoma (PCG)-causing mutants have altered metabolic activity, and correlate these to structural changes in the molecule. Methods CYP1B1.1 cDNA was mutated to four forms found in individuals with the PCG phenotype, Y81N, E229K, A330F, and R368H. Expression and stability of the mutant hemoproteins and their ability to metabolize &bgr;-estradiol, arachidonic acid, and retinoids, were determined. Alterations in mutant properties were related to structural changes by in silico examination, on the basis of the CYP1A2 crystal structure. Results CYP1B1 mutations strongly affected the stability, ease of heterologous expression, and enzymatic properties of the protein. These were related to the location of the amino acid substitutions in the CYP1B1 structure. Three of the mutations involve residues located on the surface of CYP1B1, Y81N, and E229K near the distal surface, and R368H near the proximal surface. The former two substitutions, Y81N and E229K, caused greatly reduced stability at 4°C. Y81N severely inhibited all substrate turnover, but E229K only inhibited arachidonate turnover and exhibited minimal effect on efficiency of retinoid metabolism and estradiol metabolism. The R368H mutation is relatively conservative, affecting charge-pairing with the deeper-located D374, but it severely inhibited metabolism of all substrates tested, and, like Y81N, expression of the enzyme is less facile than CYP1B1wt. The A330F mutation replaces a small alanine by a bulky phenylalanine in the enzyme active site and had major impact on substrate binding, turnover, uncoupling, and metabolite pattern. Conclusion Consistent with the hypothesis, these PCG-related mutations cause identifiable structural changes negatively impacting CYP1B1 biochemistry and stability.

Phosphorylation of cytochrome P450: regulation by cytochrome b5.

Rabbit liver cytochrome P450 LM2 and several forms of rat liver cytochrome P450 are phosphorylated by cAMP-dependent protein kinase (PKA) and by protein kinase C. Under aqueous assay conditions at neutral pH LM2 is phosphorylated only to a maximum extent of about 20 mol% by PKA. We show that detergents or alkaline pH greatly enhance the extent of phosphorylation of the cytochrome P450 substrates of cAMP-dependent protein kinase. In the presence of 0.05% Emulgen, PBRLM5, which appears to be the best cytochrome P450 substrate for cAMP-dependent protein kinase, incorporates phosphate up to about 84 mol% of enzyme. We reported previously (I. Jansson et al. (1987) Arch. Biochem. Biophys. 259, 441-448) that cytochrome b5 inhibits the phosphorylation of LM2 by cAMP-dependent protein kinase. In this paper, using PBRLM5, we demonstrate, by analysis of initial rates, that the inhibition of phosphorylation by cytochrome b5 is competitive, with a Ki = 0.48 microM. We also show that a number of forms of cytochrome P450 can be phosphorylated by protein kinase C, and that the phosphorylation of these forms by protein kinase C is also inhibited by cytochrome b5. These data suggest that the phosphorylation site(s) of cytochromes P450 may be located within or overlap the cytochrome b5 binding domain of the enzymes.