Probing the differences between rat liver outer mitochondrial membrane cytochrome b5 and microsomal cytochromes b5

Two distinct forms of cytochrome b5 exist in the rat hepatocyte. One is associated with the membrane of the endoplasmic reticulum (microsomal, or Mc, cyt b5) while the other is associated with the outer membrane of liver mitochondria (OM cyt b5). Rat OM cyt b5, the only OM cyt b5 identified so far, has a significantly more negative reduction potential and is substantially more stable toward chemical and thermal denaturation than Mc cytochromes b5. In addition, hemin is kinetically trapped in rat OM cyt b5 but not in the Mc proteins. As a result, no transfer of hemin from rat OM cyt b5 to apomyoglobin is observed at pH values as low as 5.2, nor can the thermodyamically favored ratio of hemin orientational isomers be achieved under physiologically relevant conditions. These differences are striking given the similarity of the respective protein folds. A combined theoretical and experimental study has been conducted in order to probe the structural basis behind the remarkably different properties of rat OM and Mc cytochromes b5. Molecular dynamics (MD) simulations starting from the crystal structure of bovine Mc cyt b5 revealed a conformational change that exposes several internal residues to the aqueous environment. The new conformation is equivalent to the "cleft-opened" intermediate observed in a previously reported MD simulation of bovine Mc cyt b5 [Storch, E. M., and Daggett, V. (1995) Biochemistry 34, 9682-9693]. The rat OM protein does not adopt a comparable conformation in MD simulations, thus restricting access of water to the protein interior. Subsequent comparisons of the protein sequences and structures suggested that an extended hydrophobic network encompassing the side chains of Ala-18, Ile-32, Leu-36, and Leu-47 might contribute to the inability of rat OM cyt b5 to adopt the cleft-opened conformation and, hence, stabilize its fold relative to the Mc isoforms. A corresponding network is not present in bovine Mc cyt b5 because positions 18, 32, and 47, are occupied by Ser, Leu, and Arg, respectively. To probe the roles played by Ala-18, Ile-32, and Leu-47 in endowing rat OM cyt b5 with its unusual structural properties, we have replaced them with the corresponding residues in bovine Mc cyt b5. Hence, the I32L (single), A18S/L47R (double), and A18S/L47R/I32L (triple) mutants of rat OM cyt b5 were prepared. The stability of these proteins was found to decrease in the following order: WT rat OM > rat OM I32L > rat OM A18S/L47R > rat OM A18S/L47R/I32L > bovine Mc cyt b5. The decrease in stability of the rat OM protein correlates with the extent to which the hydrophobic cluster involving the side chains of residues 18, 32, 36, and 47 has been disrupted. Complete disruption of the hydrophobic network in the triple mutant is confirmed in a 2.0 A resolution crystal structure of the protein. Disruption of the hydrophobic network also facilitates hemin loss at pH 5.2 for the double and triple mutants, with the less stable triple mutant exhibiting the greater rate of hemin transfer to apomyoglobin. Finally, 1H NMR spectroscopy and side-by-side comparisons of the crystal structures of bovine Mc, rat OM, and rat OM A18S/L47R/I32L cyt b5 allowed us to conclude that the nature of residue 32 plays a key role in controlling the relative stability of hemin orientational isomers A and B in rat OM cyt b5. A similar analysis led to the conclusion that Leu-70 and Ser-71 play a pivotal role in stabilizing isomer A relative to isomer B in Mc cytochromes b5.     

Molecular dynamics simulation studies of cytochrome b5 from outer mitochondrial and microsomal membrane

Two forms of cytochrome b(5) have been identified, associated with the outer membrane of liver mitochondria (OM cyt b(5)) and with the membrane of the endoplasmic reticulum (microsomal, Mc cyt b(5)). These proteins have very similar structures, but differ significantly in physical properties, with the OM cyt b(5) exhibiting a more negative reduction potential, higher stability, and stronger interactions with the heme. We perform molecular dynamics simulations to probe the structures and fluctuations of the two proteins in solution, to help explain the observed physical differences. We find that the structures of the two proteins, highly similar in the crystal, differ in position of a surface loop involving residues 49-51 in solution. Hydrophobic residues Ala-18, Ile-32, Leu-36, and Leu-47 tend to cluster together on the surface of rat OM cyt b(5), blocking water access to the protein interior. In bovine Mc cyt b(5), two of these positions, Ser-18 and Arg-47, are occupied by hydrophilic residues. This leads to breaking the hydrophobic cluster and allowing the protein to occupy a more open conformation. A measure of this structural transition is the opening of a cleft on the protein surface, which is 5 A wider in the OM cyt b(5) simulation compared to the Mc form. The OM protein also appears to have a more compact hydrophobic core in its beta-sheet region. These effects may be used to explain observed stability differences between the two proteins.     

Interaction of purified microsomal cytochrome P-450 with cytochrome b5

The interactions between purified microsomal cytochrome P-450 and cytochrome b₅ has been demonstrated by aqueous two-phase partition technique. Major forms of cytochrome P-450 induced by phenobarbital (P-450_LM2) and β-naphthoflavone (P-450_LM4) are almost exclusively distributed in the dextran-rich bottom phase (partition coefficient, K = 0.06), whereas NADPH-cytochrome P-450 reductase and cytochrome b₅ are mainly distributed in the polyethylene glycol-rich top phase (K = 3.5 and 2.5, respectively), when these enzymes were partitioned separately in the dextran-polyethylene glycol two-phase system. The mixing of P-450_LM with cytochrome b₅ changes the partition coefficients of both P-450_LM and cytochrome b₅ indicating that molecular interaction between P-450_LM and cytochrome b₅ occurred. Complex formation was also confirmed by optical absorbance difference spectral titration, and the stimulation of the P-450_LM-dependent 7-ethoxycoumarin and p-nitrophenetole O-deethylase activities by equal molar quantity of detergent-solubilized cytochrome b₅, but not trypsin-solubilized enzyme, in the reconstituted system. Cytochrome b₅ decreases the K_m's of both substrates for P-450_LM2-dependent O-deethylations and increases the V's of both reactions by two- to three-fold. This stimulatory effect requires the presence of phospholipid in the reconstituted enzyme system. These results suggest that cytochrome b₅ plays a role in some reconstituted drug oxidation enzyme systems and that molecular interactions among cytochrome P-450, reductase, and cytochrome b₅ are catalytically competent in the electron transport reactions.     

Defining the in Vivo Role for cytochrome b5 in cytochrome P450 function through the conditional hepatic deletion of microsomal cytochrome b5

In vitro, cytochrome b5 modulates the rate of cytochrome P450-dependent mono-oxygenation reactions. However, the role of this enzyme in determining drug pharmacokinetics in vivo and the consequential effects on drug absorption distribution, metabolism, excretion, and toxicity are unclear. In order to resolve this issue, we have carried out the conditional deletion of microsomal cytochrome b5 in the liver to create the hepatic microsomal cytochrome b5 null mouse. These mice develop and breed normally and have no overt phenotype. In vitro studies using a range of substrates for different P450 enzymes showed that in hepatic microsomal cytochrome b5 null NADH-mediated metabolism was essentially abolished for most substrates, and the NADPH-dependent metabolism of many substrates was reduced by 50-90%. This reduction in metabolism was also reflected in the in vivo elimination profiles of several drugs, including midazolam, metoprolol, and tolbutamide. In the case of chlorzoxazone, elimination was essentially unchanged. For some drugs, the pharmacokinetics were also markedly altered; for example, when administered orally, the maximum plasma concentration for midazolam was increased by 2.5-fold, and the clearance decreased by 3.6-fold in hepatic microsomal cytochrome b5 null mice. These data indicate that microsomal cytochrome b5 can play a major role in the in vivo metabolism of certain drugs and chemicals but in a P450- and substrate-dependent manner.     

Ribosomes mask cytochrome b5 on rough microsomal vesicles

Cytochrome b₅ is unmasked on the removal of ribosomes by chemical degranulation of rat liver microsomes. Reattachment of ribosomes to stripped membranes remasks this enzyme on the membrane surface. This haemoprotein may be involved either in the attachment of ribosomes to reticular membranes or in protein biosynthesis by membrane-bound ribosomes. © 1998 John Wiley & Sons, Ltd.     

The role of cytochrome b5 in adrenal microsomal steroidogenesis.

The role of cytochrome b5 in adrenal microsomal steroidogenesis was studied in guinea pig adrenal microsomes and also in the liposomal system containing purified cytochrome P-450s and NADPH-cytochrome P-450 reductase. Preincubation of the microsomes with anti-cytochrome b5 immunoglobulin decreased both 17 alpha- and 21-hydroxylase activity in the microsomes. In liposomes containing NADPH-cytochrome P-450 reductase and P-450C21 or P-450(17) alpha,lyase, addition of a small amount of cytochrome b5 stimulated the hydroxylase activity while a large amount of cytochrome b5 suppressed the hydroxylase activity. The effect of cytochrome b5 on the rates of the first electron transfer to P-450C21 in liposome membranes was determined from stopped flow measurements and that of the second electron transfer was estimated from the oxygenated difference spectra in the steady state. It was indicated that a small amount of cytochrome b5 activated the hydroxylase activity by supplying additional second electrons to oxygenated P-450C21 in the liposomes while a large amount of cytochrome b5 might suppress the activity through the interferences in the interaction between the reductase and P-450C21.     

Redox properties of microsomal reduced nicotinamide adenine dinucleotide-cytochrome b5 reductase and cytochrome b5.

Hepatic NADH-cytochrome b5 reductase was reduced by 1 mol of dithionite or NADH per mol of enzyme-bound FAD, without forming a stable semiquinone or intermediate during the titrations. However, the addition of NAD+ to the partially reduced enzyme or illumination in the presence of both NAD+ and EDTA yielded a new intermediate. The intermediate had an absorption band at 375 nm and the optical spectrum resembled anionic semiquinones seen on reduction of other flavin enzymes. Electron paramagnetic resonance measurements confirmed the free-radical nature of the species. To explain the results, a disproportionation reaction between the oxidized and reduced NAD+ complexes (E-FAD-NAD+ + E-FADH2-NAD+ in equilibrium 2E-FADH.-NAD+) is assumed. Potentiometric titration of NADH-cytochrome b5 reductase at pH 7.0 with dithionite gave a midpoint potential of -258 mV; titration with NADH gave -160 mV. This difference may be due to a difference in the relative affinity of NAD+ for the reduced and oxidized forms of the enzyme. The effects of pH on the midpoint potential of the NAD+-free enzyme were very similar to those which have been measured with free FAD. At pH 7.0, midpoint potentials of trypsin-solubilized and detergent-solubilized cytochrome b5 were 13 and 0 mV, respectively.     

Affinity chromatography of microsomal enzymes on immobilized detergent-solubilized cytochrome b5

Highly selective chromatography of microsomal enzymes has been carried out on columns of immobilized cytochrome b5 that was obtained by detergent solubilization (d-b5) of the complete amphipathic molecule. Several partially purified isozymes of cytochrome P-450 are resolved on d-b5 columns, and one high-affinity isozyme has been readily purified to homogeneity. Chromatographic selectivity and correlation of elution order of isozymes of cytochrome P-450 with direct spectral measurements of affinity constants suggests affinity chromatography on d-b5 columns. Substantial one-step enrichments of NADH-cytochrome-b5 reductase and an unstable cytochrome b5-dependent oxidase of cholesterol synthesis, 4-methyl sterol oxidase, have been obtained on d-b5 columns which further supports this conclusion. Comparison of chromatographic behavior on columns of immobilized cytochrome b5 that was obtained by trypsin solubilization (t-b5) with d-b5 columns shows marked differences which must be attributed to the absence of the hydrophobic domain of the t-b5 molecule. NADH-cytochrome-b5 reductase and the high affinity isozyme of cytochrome P-450 purified by d-b5 affinity chromatography are poorly retained on t-b5 columns. A different cytochrome P-450 isozyme with lower affinity for cytochrome b5 is only retained on d-b5 columns. Cytochrome-P-450 reductase is not retained on either column. Because affinity chromatography is suggested on d-b5 columns, the procedure may be generally applicable for predicting protein-protein interactions of microsomal electron transport components that either donate electrons to, or receive electrons from, cytochrome b5. In addition, the procedure should have considerable utilitarian application for enzyme enrichment.     

Synthesis of rat liver microsomal cytochrome b5 by free ribosomes

Free and membrane-bound polyribosomes were separated from liver homogenates and characterized by electron microscopy. Using the wheat germ cell-free translation system, total translation products of poly A+RNA extracted from free polyribosomes (poly A+RNAf) showed some correlation to total liver cytosol proteins. In contrast, translation products of poly A+RNA from membrane-bound polyribosomes (poly A+RNAmb) showed some similarity to rat serum. Antibody to purified rat serum albumin immunoprecipitated from only the translation products of poly A+RNAmb a single polypeptide of mol wt 68,000. i.e., 3,000 greater than secreted serum albumin. In contrast, antibody to detergent-extracted cytochrome b5 immunoprecipitated from only the translation products of poly A+RNAf a single polypeptide of mol wt 17,500, identical to that of microsomal cytochrome b5. A consideration of the known properties of cytochrome b5 is consistent with an exclusive site of synthesis on free ribosomes.     

Effect of n-octylamine on the reduction of mammalian hepatic microsomal cytochrome b5

1. In a preceding paper evidence was presented for the endogenous reduction of NAD(P)+ by mammalian hepatic microsomes and the concomitant reduction of cytochrome b5. The experiments reported here demonstrate that low concentrations of n-octylamine, in the presence of limiting quantities of NAD+, cause an increased level of cytochrome b5 reduction by mouse hepatic microsomes and also delays its reoxidation. 2. These effects are both NAD+ and n-octylamine dependent and appear to be due to an activation of the microsomal enzyme causing endogenous reduction of NAD(P)+ and also, in part, to inhibition of the autooxidation of reduced cytochrome b5. 3. Protection from the inhibitory action of sulfhydryl reagents on NADH-cytochrome b5 reductase was also observed in the presence of n-octylamine. 4. The results suggest that the enzyme(s) involved in the endogenous reduction of NAD(P)+ is not the microsomal alcohol dehydrogenase.     

Structural and thermodynamic encoding in the sequence of rat microsomal cytochrome b(5)

The water-soluble domain of rat microsomal cytochrome b(5) is a convenient protein with which to inspect the connection between amino acid sequence and thermodynamic properties. In the absence of its single heme cofactor, cytochrome b(5) contains a partially folded stretch of 30 residues. This region is recognized as prone to disorder by programs that analyze primary structures for such intrinsic features. The cytochrome was subjected to amino acid replacements in the folded core (I12A), in the portion that refolds only when in contact with the heme group (N57P), and in both (F35H/H39A/L46Y). Despite the difficulties associated with measuring thermodynamic quantities for the heme-bound species, it was possible to rationalize the energetic consequences of both types of replacements and test a simple equation relating apoprotein and holoprotein stability. In addition, a phenomenological relationship between the change in T(m) (the temperature at the midpoint of the thermal transition) and the change in thermodynamic stability determined by chemical denaturation was observed that could be used to extend the interpretation of incomplete holoprotein stability data. Structural information was obtained by nuclear magnetic resonance spectroscopy toward an atomic-level analysis of the effects.     

Immunochemical evidence for the participation of cytochrome b5 in microsomal stearyl-CoA desaturation reaction

A rabbit antiserum was prepared against rat liver microsomal cytochrome b₅, and utilized in demonstrating the participation of this cytochrome in the microsomal stearyl-CoA desaturation reaction. The antiserum inhibited the NADH-cytochrome c reductase activity of rat liver microsorncs, but it did not inhibit either NADH-ferricyanide or NADPH-cytochrome c reductase activity of the microsomes. Thus, the inhibitory effect of the antiserum on the microsomal electron-transferring reactions seemed to be specific to those which require the participation of cytochrome b₅.The NADH-dependent and NADPH-dependent desaturations of stearyl CoA by rat liver microsomes were strongly inhibited by the antiserum. The reduction of cytochrome b₅ by NADH-cytochrome b₅ reductase as well as the reoxidation of the reduced cytochrome b₃ by the desaturase, the terminal cyanide-sensitive factor of the desaturation system, was also strongly inhibited by the antiserum. When about 90%, of cytochrome b₅ was removed from rat liver microsomes by protease treatment, the desaturation activity of the microsomes became much more sensitive to inhibition by the antiserum. These results confirmed our previous conclusion that the reducing equivalent for the desaturation reaction is transferred from NAD(P)H to the cyanidesensitive factor mainly via cytochrome b₅ in the microsomal membranes.