Calculation of the electronic structure and spectra of model cytochrome P450 compound I.

The electronic structure and spectra of the oxyferryl (Fe=O) compound I P450 heme species, the transient putative active intermediate of cytochrome P450s, have been calculated employing a full protoporphyrin IX heme model representation. The principal aim of this work was to compare the computed spectra of this species with the observed transient spectra attributed to it. Computations were made using both nonlocal density functional theory (DFT) and semiempirical INDO/CI methods to characterize the electronic structure of the compound I P450 species. Both methods resulted in a similar antiferromagnetic doublet as the ground state with a ferromagnetic quartet excited state partner, slightly higher in energy. The INDO/ROHF/CI semiempirical method was used to calculate the spectrum of the protoporphyrin IX P450 compound I heme species in its lowest energy antiferromagnetic doublet state at the DFT optimized geometry. As a reference, the spectrum of the ferric resting form of the protoporphyrin IX P450 heme species was also calculated. The computed shifts in the Soret and Q bands of compound I relative to the resting state were both in good agreement with the corresponding experimentally observed shifts in the transient spectra of cytochrome P450cam (Biochem. Biophys. Res. Commun. 201 (1994) 1464) and chloroperoxidase (Biochem. Biophys. Res. Commun. 94 (1980) 1123) both ascribed to their common compound I heme site. This consistency provides additional, independent support for the assignment of compound I as the origin of the reported observed transient spectra.

Overexpression and purification of the membrane-bound cytochrome P450 2B4.

Expression of the membrane-bound cytochrome P450 2B4 by the pLW01-P450 expression vector, which utilizes a T7 promoter, is markedly improved by employing Escherichia coli strain C41(DE3) [Miroux, B., and Walker, J. (1996) J. Mol. Biol 260, 289--298; Bridges, A., Gruenke, L., Chang, Y.-T., Vasker, I., Loew, G., and Waskell, L. (1998) J. Biol. Chem. 273, 17036--17049]. Using this expression system, it was possible to routinely obtain an average of 50--60 mg and as high as 100 mg of cyt P450 2B4 per liter of cell culture in volumes of 500 ml. An improved purification procedure for cyt P450 2B4 is also described which allows recovery of 30% of the expressed protein. It was possible in one step using B-PER reagent and polyoxyethylene-9-lauryl ether to both lyse the E. coli and solubilize the expressed cyt P450. Cyt P450 2B4 with a specific content of 17 nmol/mg protein and a single band on polyacrylamide gel electrophoresis was routinely isolated. The yield of cyt P450 from the improved purification procedure is twice that from the original procedure and the purity of the recovered protein typically has a specific content of 17 nmol cyt P450/mg of protein.

NMR studies of the association of cytochrome b5 with cytochrome c.

In an effort to gain greater insight into the molecular mechanism of the electron-transfer reactions of cytochrome b(5), the bovine cytochrome b(5)-horse cytochrome c complex has been investigated by high-resolution multidimensional NMR spectroscopy using (13)C, (15)N-labeled cytochrome b(5) expressed from a synthetic gene. Chemical shifts of the backbone (15)N, (1)H, and (13)C resonances for 81 of the 82 residues of [U-90% (13)C,U-90% (15)N]-ferrous cytochrome b(5) in a 1:1 complex with ferrous cytochrome c were compared with those of ferrous cytochrome b(5) in the absence of cytochrome c. A total of 51% of these residues showed small, but significant, changes in chemical shifts (the largest shifts were 0.1 ppm for the amide (1)H, 1.15 for (13)C(alpha), 1.03 ppm for the amide (15)N, and 0.15 ppm for the (1)H(alpha) resonances). Some of the residues exhibiting chemical shift changes are located in a region that has been implicated as the binding surface to cyt c [Salemme, F. R. (1976) J. Mol. Biol. 10, 563-568]. Surprisingly, many of the residues with changes are not located on this surface. Instead, they are located within and around a cleft observed to form in a molecular dynamics study of cytochrome b(5) [Storch, E. M., and Daggett, V. (1995) Biochemistry 34, 9682-9693](.) The rim of this cleft can readily accommodate cytochrome c. Molecular dynamics simulations of the Salemme and cleft complexes were performed for 2 ns and both complexes were stable.

High-level expression in Escherichia coli and purification of the membrane-bound form of cytochrome b(5).

Expression of the membrane-bound form of rabbit cytochrome b(5) in Escherichia coli has been significantly improved through the use of the T7 expression vector pLW01 (A. Bridges, L. Gruenke, Y.-T. Chang, I. Vakser, G. Loew, and L. Waskell, 1998, J. Biol. Chem. 273, 17036-17049) in conjunction with strain C41(DE3) (B. Miroux and J. Walker, 1996, J. Mol. Biol. 260, 289-298). Cell cultures expressing the cytochrome b(5) contained an average of 820 mg/liter of culture and reached peak levels as high as 1100 mg/liter when higher antibiotic concentrations were used. Maximal levels were obtained from cultures when expression was induced with 10 microM IPTG. Approximately 90% of the cytochrome b(5) was expressed as apoprotein which was reconstituted by addition of exogenous heme. The cytochrome b(5) was purified from detergent-solubilized bacterial membranes using anion-exchange chromatography on DEAE-Sepharose followed by size-exclusion chromatography on Superdex-75. Purification of cytochrome b(5) from a 500-ml culture yielded 121 mg of protein which had a specific content of 50 nmol of heme per milligram of protein with an overall recovery of 35%. The final cytochrome b(5) was free of any detectable contaminants when analyzed by SDS-PAGE.

Identification of the binding site on cytochrome P450 2B4 for cytochrome b5 and cytochrome P450 reductase.

A model of cytochrome P450 2B4, which was constructed by homology modeling with the four known crystal structures of the cytochromes P450 (Chang, T.-T., Stiffelman, O. B., Vakser, I. A., Loew, G. H., Bridges, A., and Waskell, L. (1997) Protein Eng. 10, 119-129), was used to select amino acids predicted, by computer docking studies and numerous previous biochemical and site-directed mutagenesis studies, to be involved in binding the heme domain of cytochrome b5. Twenty-four amino acid residues located on both the distal and the proximal surface of the molecule were chosen for mutagenesis. These 24 mutant proteins were expressed in Escherichia coli, purified, and characterized with respect to their ability to bind cytochrome b5 and support substrate oxidation. Seven mutants, R122A, R126A, R133A, F135A, M137A, K139A, and K433A, all on the proximal surface of cytochrome P450 2B4 near the heme ligand, were identified that exhibited decreased ability to bind cytochrome b5. All of the mutants except K433A are located in either the C or C* helices or their termini. In addition, these seven mutants and two additional mutants on the proximal surface of cytochrome P450, R422A and R443A, were shown to exhibit decreased binding to cytochrome P450 reductase. These studies indicate that the binding sites for cytochrome b5 and cytochrome P450 reductase are, as predicted, located on the proximal surface of cytochrome P450 2B4 and are partially overlapping but not identical.

Resonance Raman spectral properties and stability of manganese protoporphyrin IX cytochrome b5.

The structure and stability of cytochrome b5 reconstituted with manganese protoporphyrin IX instead of iron protoporphyrin IX has been investigated by resonance Raman spectroscopy and stopped-flow visible spectroscopy. The resonance Raman spectrum of MnIII cytochrome b5 was consistent with a high-spin hexacoordinate MnIII protoporphyrin IX structure that converted to a high-spin pentacoordinate structure at higher laser power. The resonance Raman spectrum of MnII cytochrome b5 indicated a high-spin pentacoordinate structure which was independent of laser power. Studies of the binding of MnIII protoporphyrin IX to apocytochrome b5 indicated that the MnIII-containing porphyrin bound much less tightly to the protein than did heme. Although the second-order rate constant at 20 degrees C for the association of heme with apocytochrome b5 (4.5 x 10(7) M(-1) s(-1)) was estimated to be only 1 order of magnitude higher than that with Mn protoporphyrin IX (3.3 x 10(6) M(-1) s(-1)), the dissociation of manganese substituted cytochrome b5 into the apoprotein and free Mn protoporphyrin IX occurs with a first-order rate constant of 1.2 x 10(-2) s(-1) at 20 degrees C while the dissociation of heme from cytochrome b5 at room temperature occurs 3 orders of magnitude more slowly with a first-order rate constant of 1.67 x 10(-5) s(-1) [Vergeres, G., Chen, D. Y., Wu, F.F., & Waskell, L. (1993) Arch. Biochem. Biophys. 305, 231-241]. The equilibrium dissociation constant for manganese-substituted cytochrome b5 increased with temperature from 4 nM at 20 degrees C to 14 nM at 37 degrees C. These results suggest that, in the reconstituted cytochrome P450 metabolizing system, especially in studies done with low protein concentrations (0.1 microM), and at elevated temperatures (37 degrees C), as much as 30% of the manganese-substituted cytochrome b5 may dissociate to free Mn-protoporphyrin IX and apocytochrome b5.

Construction of a 3D model of cytochrome P450 2B4.

A three-dimensional structural model of rabbit phenobarbital-inducible cytochrome P450 2B4 (LM2) was constructed by homology modeling techniques previously developed for building and evaluating a 3D model of the cytochrome P450choP isozyme. Four templates with known crystal structures including cytochrome P450cam, terp, BM-3 and eryF were used in multiple sequence alignments and construction of the cytochrome P450 2B4 coordinates. The model was evaluated for its overall quality using available protein analysis programs and found to be satisfactory. The model structure was stable at room temperature during a 140 ps unconstrained full protein molecular dynamics simulation. A putative substrate access channel and binding site were identified. Two different substrates, benzphetamine and androstenedione, that are metabolized by cytochrome P450 2B4 with pronounced product specificity were docked into the putative binding site. Two orientations were found for each substrate that could lead to the observed preferred products. Using a geometric fit method three regions on the surface of the model cytochrome P450 structure were identified as possible sites for interaction with cytochrome b5, a redox partner of P450 2B4. Residues that may interact with the substrates and with cytochrome b5 have been identified and mutagenesis studies are currently in progress.

Pseudocontact shifts used in the restraint of the solution structures of electron transfer complexes.

The geometry of the ferricytochrome b5-ferricytochrome c complex has been analysed using long-range interprotein paramagnetic dipolar shifts. Heteronuclear filtered NMR spectra of samples containing 15N-labelled cytochrome b5 in complex with unlabelled cytochrome c allowed unambiguous assessment of pseudocontact shifts relative to diamagnetic reference states. Because pseudocontact shifts can be observed for protons as much as 20 A from the paramagnetic centre, this approach allows study of electron transfer proteins in fast exchange. Our findings provide the first physical evidence confirming hypotheses presented in previous theoretical studies. This absence of certain predicted shifts that are expected based on the best fit to a static model of the complex suggests that cytochrome b5 is more dynamic in solution than in the crystal, in agreement with molecular dynamics simulations.

The stoichiometry of the cytochrome P-450-catalyzed metabolism of methoxyflurane and benzphetamine in the presence and absence of cytochrome b5.

The complete stoichiometry of the metabolism of the cytochrome b5 (cyt b5)-requiring substrate, methoxyflurane, by purified cytochrome P-450 2B4 was compared to that of another substrate, benzphetamine, which does not require cyt b5 for its metabolism. Cyt b5 invariably improved the efficiency of product formation. That is, in the presence of cyt b5 a greater percentage of the reducing equivalents from NADPH were utilized to generate substrate metabolites, primarily at the expense of the side product, superoxide. With methoxyflurane, cyt b5 addition always resulted in an increased rate of product formation, while with benzphetamine the rate of product formation remained unchanged, increased or decreased. The apparently contradictory observations of increased reaction efficiency but decrease in total product formation for benzphetamine can be explained by a second effect of cyt b5. Under some experimental conditions cyt b5 inhibits total NADPH consumption. Whether stimulation, inhibition, or no change in product formation is observed in the presence of cyt b5 depends on the net effect of the stimulatory and inhibitory effects of cyt b5. When total NADPH consumption is inhibited by cyt b5, the rapidly metabolized, highly coupled (approximately equal to 50%) substrate, benzphetamine, undergoes a net decrease in metabolism not counterbalanced by the increase in the efficiency (2-20%) of the reaction. In contrast, in the presence of the slowly metabolized, poorly coupled (approximately equal to 0.5-3%) substrate, methoxyflurane, inhibition of total NADPH consumption by cyt b5 was never sufficient to overcome the stimulation of product formation due to an increase in efficiency of the reaction.

The carboxyl terminus of the membrane-binding domain of cytochrome b5 spans the bilayer of the endoplasmic reticulum.

Preliminary studies (Vergères, G., and Waskell, L. (1992) J. Biol. Chem. 267, 12583-12591) have suggested that the carboxyl-terminal membrane-binding domain of cytochrome b5 traverses the membrane and that the carboxyl terminus is in the lumen of the endoplasmic reticulum. In order to confirm and extend these studies, additional experiments were conducted. The gene coding for rat cytochrome b5 was transcribed and the resulting mRNA was translated in vitro in a rabbit reticulocyte lysate in the presence of microsomes. The binding and topology of cytochrome b5 were investigated by treating microsomes containing the newly incorporated cytochrome b5 with carboxypeptidase Y and trypsin. Our studies indicate that cytochrome b5 is inserted both co- and post-translationally into microsomes in a topology in which the membrane-binding domain spans the bilayer with its COOH terminus in the lumen. Cytochrome b5 is also incorporated into microsomes pretreated with trypsin in a topology indistinguishable from the one resulting from the insertion of the protein into untreated microsomes, reconfirming that cytochrome b5 does not use the signal recognition particle-dependent translocation machinery. Our results do not allow a distinction to be made between a spontaneous insertion mode or some other trypsin-resistant receptor-mediated mechanism. A role for Pro115 in the middle of the membrane-binding domain of cytochrome b5 was also examined by mutating it to an alanine and subsequently characterizing the ability of the mutant protein to be incorporated into membranes. The mutant protein inserted more slowly in vitro into microsomes as well as into pure lipid bilayers by a factor of 2 to 3.

Cytochrome b5, its functions, structure and membrane topology.

The first part of the present communication reviews recent advances in our understanding of the known physiological functions of cytochrome b5. In addition, one section is devoted to a description of a recently discovered function of cytochrome b5, namely its involvement in the synthesis of the oncofetal antigen N-glycolylneuraminic acid. The second part of the article summarizes site-directed mutagenesis studies, primarily conducted in the author's laboratory, in both the catalytic heme-binding and membrane-binding domain of cytochrome b5. These studies have shown that: 1) the membrane binding domain of cytochrome b5 spans the bilayer; 2) cytochrome b5 lacking 19 COOH-terminal amino acids does not bind to membrane bilayers; and 3) specific amino acids in the membrane binding domain have been mutated and shown not to be essential for the function of cytochrome b5 with its redox partners.

Investigation of the rate limiting step for electron transfer from NADPH:cytochrome P450 reductase to cytochrome b5: a laser flash-photolysis study.

The reduction kinetics of the one-electron-reduced cytochrome P450 reductase:cytochrome b5 complex (P450R1e:b5ox) has been investigated by the laser flash-photolysis technique, using the semiquinone of 5-deazariboflavin (5-dRfH.) as the reductant. Investigation of the kinetic properties of the individual components at 470 nm indicated that P450R1e and b5ox were reduced via second-order kinetics by 5-dRfH. with rate constants of 1 x 10(8) M-1 s-1 and 4.2 x 10(8) M-1 s-1, respectively. Intramolecular electron transfer from (laser reduced) FADH. to FMNH. was measured at 585 nm and a first-order rate constant of 36 s-1 was obtained for this process. Reduction of the preformed P450R1e:b5ox complex by 5-dRfH. was biphasic and second-order rate constants of 5.4 x 10(8) M-1 s-1 and 7.5 x 10(7) M-1 s-1 were obtained for the fast and slow phases of reduction. The time-resolved flash-induced difference spectrum was consistent with the simultaneous direct reduction (by 5-dRfH.) of protein-bound flavin and heme, followed by an additional slower first-order intracomplex electron transfer (kLim = 37 s-1) from protein-bound flavin semiquinone to heme. The results indicate that the (laser-generated) two-electron-reduced form of cytochrome P450 reductase is catalytically competent in the transfer of reducing equivalents to oxidized cytochrome b5 and suggest that formation of FMNH2 as a result of internal electron transfer from FADH. to FMNH. within P450R1e is the rate limiting step in the reduction of cytochrome b5 by cytochrome P450 reductase. The Kd of the cytochrome P450 reductase:cytochrome b5 complex was estimated to be approximately 1.5 x 10(-6) M.

Kinetics of the reduction of cytochrome b5 with mutations in its membrane-binding domain.

In an attempt to understand which amino acids in the membrane anchor of cytochrome b5 might be determinants of its ability to support the cytochrome P450-catalyzed oxidation of selected substrates, the synthetic rat cytochrome b5 gene has been mutated by site-directed mutagenesis. The mutant proteins have been expressed in Saccharomyces cerevisiae, purified and assayed for their ability to support the cytochrome P450-catalyzed metabolism of the cytochrome b5 requiring substrate methoxyflurane (G. Vergères and L. Waskell, 1992, J. Biol. Chem. 267, 12583-12591). The rate of reduction of the cytochromes b5 by cytochrome P450 reductase has been examined by stopped-flow spectrophotometry to determine whether an altered rate of reduction of cytochrome b5 could explain the observed activity of cytochrome b5 in the purified reconstituted mixed-function oxidase system. A mutant in which the 22-amino-acid membrane anchor was replaced by a sequence of 22 leucines was unable to support methoxyflurane metabolism in the reconstituted system and was reduced by cytochrome P450 reductase at a rate (k = 4.5 x 10(-3) s-1) slow enough to explain this finding. Comparison of the rate of reduction of this mutant cytochrome b5 in 0.025% Tergitol and 40 microM dilauroylphosphatidylcholine suggests that this slow rate of reduction may be explained partially by aggregation of the polyleucine protein. The Pro115Stop mutant protein, which has been truncated by 19 amino acids in its COOH terminus resulting in a protein with one-half of the putative membrane anchor, supports methoxyflurane oxidation at 12-20% of the rate of the wild type protein. In addition it is reduced by cytochrome P450 reductase at a rate which should be capable of supporting a normal rate of production formation. The fact that the Pro115Stop mutant can be reduced at a rate capable of supporting a normal rate of methoxyflurane oxidation but in fact only supports methoxyflurane oxidation at 30% of the normal rate suggests that the mutant protein is deficient in its interactions with cytochrome P450. The mutant proteins, Pro115Ala and Ala116Pro, behaved essentially as did the wild type protein demonstrating that the presence (Pro115Ala) or absence (Ala116Pro) of an alpha helix in the middle of the putative membrane-binding domain of cytochrome b5 was not a determinant of the interaction of cytochrome b5 with cytochrome P450 reductase and cytochrome P450.(ABSTRACT TRUNCATED AT 400 WORDS)

A model system for studying membrane biogenesis. Overexpression of cytochrome b5 in yeast results in marked proliferation of the intracellular membrane.

Cytochrome b5 is an amphipathic microsomal protein that is anchored to the endoplasmic reticulum by a single hydrophobic transmembrane alpha-helix located near the carboxyl terminus of the protein. In yeast, cytochrome b5 provides electrons for fatty acid desaturation and ergosterol biosynthesis. High level expression of cytochrome b5 in Saccharomyces cerevisiae was achieved using the yeast metallothionein promoter and a synthetic cytochrome b5 gene. In order to accommodate the markedly increased amount of the membrane-bound cytochrome b5, the yeast cell proliferated its nuclear membrane. As many as 20 pairs of stacked membranes could be observed to partially encircle the nucleus. This morphological arrangement of membrane around the nucleus is known as a karmella. In an effort to understand which part of the cytochrome b5 molecule, i.e. the membrane anchor or the soluble heme domain, which is competent in electron transfer, provided the signal for the de novo membrane biogenesis, a series of studies, including site-directed mutagenesis, was undertaken. The results of these experiments demonstrated that the inactive hemedeficient apo form of the membrane-bound protein stimulates membrane proliferation to the same extent as the holo wild-type protein, whereas cytosolic forms of cytochrome b5 did not induce membrane synthesis. These data demonstrate that membrane proliferation is a consequence of the cell's ability to monitor the level of membrane proteins and to compensate for alterations in these levels rather than the result of the ability of the extra cytochrome b5 to catalyze synthesis of extra lipid that had to be accommodated in new membrane. Site-directed mutagenesis studies of the membrane binding domain of cytochrome b5 provided additional clues about the nature of the signal for membrane proliferation. Replacement of the membrane anchor by a non-physiological nonsense sequence of 22 leucines gave rise to a mutant protein that triggered membrane biosynthesis. The conclusion from these experiments is clear; the signal for membrane proliferation does not reside in some specific amino acid sequence but instead in the hydrophobic properties of the proliferant. Interestingly, these membranes are somewhat diminished in quantity and have a slightly altered morphology compared to those induced by the wild-type protein. It was also observed that disruption of the putative alpha helix of the membrane anchor by an Ala116Pro mutation, which gives rise to two sequential prolines at positions 115 and 116 results in a protein with diminished capacity to induce membrane formation.(ABSTRACT TRUNCATED AT 400 WORDS)

The function of tyrosine 74 of cytochrome b5.

Tyrosine 74, which is part of a hydrophobic patch on the surface of rat cytochrome b5, also forms van der Waals contacts with the heme prosthetic group of the protein. In addition it is a member of an aromatic network of amino acids which includes Phe-35 and the axial ligand, His-39. Because of its strategic location in the protein, the Tyr-74 residue was mutated to a lysine in order to investigate how it affected the interaction of heme with the protein and whether it might be an alternative binding site and an electron transfer path which cytochrome b5 would use with its amphipathic electron transfer partners cytochrome P450 and its corresponding NADPH cytochrome P450 reductase. The mutant protein receives electrons from NADPH cytochrome P450 reductase and provides the second electron to cytochrome P450 to catalyze the metabolism of methoxyflurane, a substrate which requires cytochrome b5 for its metabolism, at the same rate as the wild type protein. The Tyr74Lys mutant exhibits a normal redox potential and spectroscopic properties identical to those of the wild type protein. Under equilibrium conditions in the presence of urea, heme dissociation and denaturation occur simultaneously with a free energy of 3.4 kcal/mol. The free energy of activation of heme dissociation from the wild type protein is 22.7 kcal/mol. The free energy and free energy of activation of the Tyr74Lys mutant are 1.4 kcal/mol less than the wild type values, indicating that the mutant binds heme 10-fold less tightly and dissociates heme 10 times faster than the wild type protein. Heme transfer experiments demonstrate that heme spontaneously dissociates 6 times faster from the mutant than the wild type protein (t1/2 = 1.9 and 11.5 h, respectively). The most likely conformation of Lys-74 in the mutant protein was determined by calculating the Lys-74 rotamer with the minimal energy using an energy-based conformation search method. This conformation was subsequently modeled on the computer graphics. Not unexpectedly, the side chain of Lys-74 is shifted toward the surface of the protein to allow solvation of the positive charge on the epsilon amino group of lysine. This movement of the lysine residue results in the formation of a cavity on the surface of the cytochrome b5 molecule, which exposes a heme methyl and vinyl group to aqueous solvent thereby destabilizing the binding between the protein and its hydrophobic prosthetic heme group.

Novel heteronuclear methods of assignment transfer from a diamagnetic to a paramagnetic protein: application to rat cytochrome b5.

15N and 1H resonance assignments for backbone and side-chain resonances of both equilibrium forms of rat ferricytochrome b5 have been obtained, using a combination of novel heteronuclear assignment transfer methods from the known assignments of the diamagnetic protein [Guiles, R. D., Basus, V. J., Kuntz, I. D., & Waskell, L. A. (1992) Biochemistry 31, 11365-11375] and computational methods which depend on an accurate determination of the orientation of the components of the susceptibility tensor. The transfer of amide proton resonance assignments takes advantage of the apparent insensitivity of amide 15N resonances to pseudocontact effects, evident in overlays of 15N-1H heteronuclear correlation spectra. Amide-proton resonance assignments tentatively transferred from the known diamagnetic assignments to the paramagnetic form of the protein were confirmed using conventional assignment strategies employing 600-MHz COSY, HOHAHA, and NOESY spectra of the oxidized protein. As was observed in rat ferrocytochrome b5, more than 40% of all residues exhibited NMR detectable heterogeneity due to the two different orientations of the heme. Complete assignment of both forms enabled accurate determination of the orientation of the susceptibility tensor for both conformations of the heme. The orientation of the z-component of the susceptibility tensors for the two forms are indistinguishable, while the in-plane components appear to differ by about 6 degrees. Differences in the orientation of the in-plane susceptibility components are undoubtedly due dominantly to the relative axial rotation of the heme of between 5 degrees and 10 degrees indicated by the NOESY contacts to the protein observed in the spectra of the ferrocytochrome [Guiles, R. D., Basus, V. J., Kuntz, I. D., & Waskell, L. A. (1992) Biochemistry 31, 11365-11375; Pochapsky, T. C., Sligar, S. G., McLachlan, S. J., & LaMar, G. N. (1990) J. Am. Chem. Soc. 112, 5258-5263].

Sequence-specific 1H and 15N resonance assignments for both equilibrium forms of the soluble heme binding domain of rat ferrocytochrome b5.

15N and 1H resonance assignments for backbone and side-chain resonances of both equilibrium forms of rat ferrocytochrome b5 have been obtained, using 15N-1H heteronuclear correlation methods employing globally 15N-labeled protein. Unlike other cytochrome b5 species assigned to date (Guiles et al., 1990) the rat cytochrome exists as an equilibrium distribution of conformers in nearly equal abundance (Lee et al., 1990). The ratio of conformers present in all other species variants is approximately 1:9. More than 40% of all residues of the rat protein exhibit NMR-detectable heterogeneity due to the 180 degrees rotation of the heme about the alpha, gamma-meso axis. NOESY and HOHAHA relayed 15N-1H double-DEPT heteronuclear correlation methods were an indispensible tool for the deconvolution of a system with this level of heterogeneity. Differences in the resonance assignments between the two equilibrium conformers were found to be as great as differences between species variants we have previously reported. On the basis of the magnitude and extent of the observed chemical shift differences and specific NOESY connectivities observed in the two isomers, we believe the two equilibrium conformers differ not only by a simple back-to-front flip of the heme but also by an additional rotation about an axis normal to the heme plane as has been previously suggested by Pochapsky et al. (1990). A short segment of the protein at the N-terminus could not be assigned, presumably due to rapid exchange of solvent-accessible amide protons in this disordered segment of the protein. Assignments for 93 of the 98 residues of this 12-kDa protein have been obtained.

Expression of cytochrome b5 in yeast and characterization of mutants of the membrane-anchoring domain.

The soluble and membrane-bound forms of the synthetic rat cytochrome b5 gene have been expressed in Saccharomyces cerevisiae. In order to examine the topology and function of the COOH-terminal membrane binding domain of cytochrome b5, mutants have been constructed, expressed, purified, and partially characterized. Pro-115 is located in the middle of the putative alpha-helical membrane-anchoring domain of cytochrome b5 and has been hypothesized to give rise to either a hairpin-like loop or approximately equal to 26 degrees kink in the helix, depending on whether it exists, respectively, in the cis or trans configuration. The Pro-115----Ala mutant, which is expected to have a straight transmembrane helix, inserted normally into the endoplasmic reticulum and exhibited wild type levels of activity in yeast microsomes and in vitro in the cytochrome P-450 mixed function oxidation system. Since a hairpin structure does not appear to be essential, it is likely that the membrane binding domain of cytochrome b5 spans the membrane. Characterization of the truncated cytochrome b5 molecule, Pro-115----Stop, lacking 19 amino acids at the COOH terminus indicates that the distal part of the membrane binding domain of cytochrome b5 is necessary for in vivo binding to the endoplasmic reticulum and for functioning with its membrane-associated electron transfer partners. Replacement of Ser-104 to Met-125, the putative membrane-anchoring domain of cytochrome b5, with 22 leucine residues results in a protein which targets to the endoplasmic reticulum but the extent of its reduction is only 50% of that of the wild type in yeast microsomes. In vitro, the polyleucine mutant is unable to support substrate oxidation by cytochrome P-450. The mutation of Ala-131 and Glu-132, amino acids flanking the transmembrane domain, to lysines resulted in a protein with normal membrane topology and function.

Fluoride metabolites after prolonged exposure of volunteers and patients to desflurane.

We examined the metabolism of desflurane in 13 healthy volunteers given 7.35 +/- 0.81 MAC-hours (mean +/- SD) of desflurane and 26 surgical patients given 3.08 +/- 1.84 MAC-hours (mean +/- SD). Markers of desflurane metabolism included fluoride ion measured via an ion-specific electrode, nonvolatile organic fluoride measured after sodium fusion of urine samples, and trifluoroacetic acid determined by a gas chromatographic-mass spectrometric method. In both volunteer and patient groups, postanesthesia serum fluoride ion concentrations did not differ from background fluoride ion concentrations. Similarly, postanesthesia urinary excretion of fluoride ion and organic fluoride in volunteers was comparable to preanesthesia excretion rates. However, small but significant levels of trifluoroacetic acid were found in both serum and urine from volunteers after exposure to desflurane. A peak serum concentration of 0.38 +/- 0.17 mumol/L of trifluoroacetic acid and a peak urinary excretion rate of 0.169 +/- 0.107 mumol/h were detected in volunteers at 24 h after desflurane exposure. Although these increases in trifluoroacetic acid after exposure to desflurane were statistically significant, they are approximately 10-fold less than levels seen after exposure to isoflurane. Thus, desflurane strongly resists biodegradation, but a small amount is metabolized in humans.