Apocytochrome B5 Research Articles

Engineering Aromatic-Aromatic Interactions To Nucleate Folding in Intrinsically Disordered Regions of Proteins.

Aromatic interactions are an important force in protein folding as they combine the stability of a hydrophobic interaction with the selectivity of a hydrogen bond. Much of our understanding of aromatic interactions comes from "bioinformatics" based analyses of protein structures and from the contribution of these interactions to stabilizing secondary structure motifs in model peptides. In this study, the structural consequences of aromatic interactions on protein folding have been explored in engineered mutants of the molten globule protein apo-cytochrome b5. Structural changes from disorder to order due to aromatic interactions in two variants of the protein, viz., WF-cytb5 and FF-cytb5, result in significant long-range secondary and tertiary structure. The results show that 54 and 52% of the residues in WF-cytb5 and FF-cytb5, respectively, occupy ordered regions versus 26% in apo-cytochrome b5. The interactions between the aromatic groups are offset-stacked and edge-to-face for the Trp-Phe and Phe-Phe mutants, respectively. Urea denaturation studies indicate that both mutants have a Cm higher than that of apo-cytochrome b5 and are more stable to chaotropic agents than apo-cytochrome b5. The introduction of these aromatic residues also results in "trimer" interactions with existing aromatic groups, reaffirming the selectivity of the aromatic interactions. These studies provide insights into the aromatic interactions that drive disorder-to-order transitions in intrinsically disordered regions of proteins and will aid in de novo protein design beyond small peptide scaffolds.

Cytochrome b5 Increases Cytochrome P450 3A4-Mediated Activation of Anticancer Drug Ellipticine to 13-Hydroxyellipticine Whose Covalent Binding to DNA Is Elevated by Sulfotransferases and N,O-Acetyltransferases

The antineoplastic alkaloid ellipticine is a prodrug, whose pharmacological efficiency is dependent on its cytochrome P450 (P450)- and/or peroxidase-mediated activation in target tissues. The P450 3A4 enzyme oxidizes ellipticine to five metabolites, mainly to 13-hydroxy- and 12-hydroxyellipticine, the metabolites responsible for the formation of ellipticine-13-ylium and ellipticine-12-ylium ions that generate covalent DNA adducts. Cytochrome b(5) alters the ratio of ellipticine metabolites formed by P450 3A4. While the amounts of the detoxication metabolites (7-hydroxy- and 9-hydroxyellipticine) were not changed with added cytochrome b(5), 12-hydroxy- and 13-hydroxyellipticine, and ellipticine N(2)-oxide increased considerably. The P450 3A4-mediated oxidation of ellipticine was significantly changed only by holo-cytochrome b(5), while apo-cytochrome b(5) without heme or Mn-cytochrome b(5) had no such effect. The change in amounts of metabolites resulted in an increased formation of covalent ellipticine-DNA adducts, one of the DNA-damaging mechanisms of ellipticine antitumor action. The amounts of 13-hydroxy- and 12-hydroxyellipticine formed by P450 3A4 were similar, but more than 7-fold higher levels of the adduct were formed by 13-hydroxyellipticine than by 12-hydroxyellipticine. The higher susceptibility of 13-hydroxyellipticine toward heterolytic dissociation to ellipticine-13-ylium in comparison to dissociation of 12-hydroxyellipticine to ellipticine-12-ylium, determined by quantum chemical calculations, explains this phenomenon. The amounts of the 13-hydroxyellipticine-derived DNA adduct significantly increased upon reaction of 13-hydroxyellipticine with either 3'-phosphoadenosine-5'-phosphosulfate or acetyl-CoA catalyzed by human sulfotransferases 1A1, 1A2, 1A3, and 2A1, or N,O-acetyltransferases 1 and 2. The calculated reaction free energies of heterolysis of the sulfate and acetate esters are by 10-17 kcal/mol more favorable than the energy of hydrolysis of 13-hydroxyellipticine, which could explain the experimental data.

Abstract 5419: The plant alkaloid ellipticine is detoxified by cytochrome P450 1A1/2 (CYP 1A1/2) but activated and forms DNA adducts if CYP1A1 or 1A2 is combined with cytochrome b5: an explanation for an in vivo/in vitro discrepancy

Abstract Ellipticine (5,11-dimethyl-6H-pyrido[4,3-b] carbazole) is an alkaloid with anti-cancer activity elicited by interfering with many cellular functions. One of these is the formation of DNA adducts upon activation by CYP or peroxidases. The metabolites responsible for detoxication or DNA adduct formation have been characterised and CYP3A1 and the othologous human enzyme CYP3A4 are the most efficient activating enzymes in microsomes from either species (Stiborová M et al. Cancer Res 64 (2004) 8374-8380). In vivo also CYP1A effectively activate ellipticine to DNA adducts, while isolated CYP1A1/2 detoxify ellipticine mainly to its 9-hydroxy derivative. Cytochrome b5 has been shown to modulate CYP catalysis by either transferring the second electron to CYPs, or by allosteric interaction thereby leading to higher CYP enzyme activity (Schenkman, J.B., I. Jansson Pharmacol. Ther. 97(2003) 139-52). Isolated cytochrome b5 was added to isolated CYP1A1 or 1A2 reconstituted with NADPH-CYP reductase in liposomes at different ratios of the three enzymes. Apo-cytochrome b5 (containing no heme) was generated in transfected E.coli (Kotrbová V. et al. Protein Expr Purif., 66(2009) 203-209) and used as such or reconstituted with heme or Mn-protoporphyrin IX. Metabolites were analysed by HPLC and UV detection and quantified with standards. DNA adducts were determined by the nuclease P1 version of the 32P-postlabeling assay. With both CYP1A1 and 1A2 the pattern of ellipticine metabolites in incubations containing cytochrome b5 shifted from detoxication to activation. This shift depended on the ratio of CYP1A to cytochrome b5 with optimal yield of 12-hydroxy- and 13-hydroxy ellipticine at a ratio of 1:5, CYP1A to cytochrome b5. The shift was more pronounced for CYP1A1 than for 1A2 leading to 6.5 fold higher DNA adduct levels than without cytochrome b5 correlating with 6 fold higher 13-hydroxy-ellipticine amounts, the metabolite responsible for the major deoxyguanosin adduct. The cytochrome b5 holo-enzyme is necessary for this effect, since neither the apo-enzyme nor the apo-enzyme reconstituted with Mn-protoporphyrin IX had the same effect. Other heme containing proteins, like myoglobin or cytochrome c had no effect. Ellipticine is, to our knowledge, the first xenobiotic compound where cytochrome b5 shifts the metabolism by CYP from detoxication to activation and DNA adduct formation. This is important also when analysing the pharmacological effects in vivo, since ellipticine induces both CYP1A and cytochrome b5. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5419. doi:10.1158/1538-7445.AM2011-5419

Folding behaviors of apocytochrome b5 and its mutants: Insights from high temperature molecular dynamics simulations

Apocytochrome b 5 (apocyt b 5) with heme removal from the heme-binding core 1, and its mutants with amino acid replaced in the hydrophobic core 2, namely apocyt b 5 Y7P, P81A and H15R/S20E, have been subjected to molecular dynamics (MD) simulation at high temperature (500 K) for elucidating their folding behaviors. The early events upon thermal induced unfolding were found to be in good agreement with available experimental results, and the lowest stability of Y7P was predicted among the four apoproteins. The influences of these key residues on protein folding behavior were compared directly at an atomic level. At the same time, the influences of non-native interactions of hydrogen bonds and salt-bridges on protein stabilities were analyzed in detail. The insights from current MD simulations are valuable for understanding the apoprotein folding and the holoprotein formation in terms of heme proteins.

Preparation of a biologically active apo-cytochrome b5via heterologous expression in Escherichia coli

Cytochrome b5 (b5) has been shown to modulate many cytochrome P450 (CYP)-dependent reactions. In order to elucidate the mechanism of such modulations, it is necessary to evaluate not only the effect of native b5 on CYP-catalyzed reactions, but also that of the apo-cytochrome b5 (apo-b5). Therefore, the apo-b5 protein was prepared using a heterologous expression in Escherichia coli. The gene for rabbit b5 was constructed from synthetic oligonucleotides using polymerase chain reaction (PCR), cloned into pUC19 plasmid and amplified in DH5α cells. The gene sequence was verified by DNA sequencing. The sequence coding b5 was cleaved from pUC19 by NdeI and XhoI restriction endonucleases and subcloned to the expression vector pET22b. This vector was used to transform E. coli BL-21 (DE3) Gold cells by heat shock. Expression of b5 was induced with isopropyl β-d-1-thiogalactopyranoside (IPTG). The b5 protein, produced predominantly in its apo-form, was purified from isolated membranes of E. coli cells by chromatography on a column of DEAE–Sepharose. Using such procedures, the homogenous preparation of apo-b5 protein was obtained. Oxidized and reduced forms of the apo-b5 reconstituted with heme exhibit the same absorbance spectra as native b5. The prepared recombinant apo-b5 reconstituted with heme can be reduced by NADPH:CYP reductase. The reconstituted apo-b5 is also fully biologically active, exhibiting the comparable stimulation effect on the CYP3A4 enzymatic activity towards oxidation of 1-phenylazo-2-hydroxynaphthalene (Sudan I) as native rabbit and human b5.

Dynamic consequences of mutating the typical HPGG motif of apocytochrome b5 revealed by computer simulation

Apocytochrome b 5 with a typical heme-binding motif of HPGG, and its variants with mutated motifs, GPGG, GPGH, HVGG, and HPGP, have been subjected to molecular dynamics simulation. Comparison of the dynamic consequences has revealed the crucial role of HPGG in assembling the heme group of cytochrome b 5 and in modulating protein structure, property and function.

Cytochrome b5 Inhibits Electron Transfer from NADPH-Cytochrome P450 Reductase to Ferric Cytochrome P450 2B4

Experiments demonstrating that cytochrome (cyt) b5 inhibits the activity of cytochrome P450 2B4 (cyt P450 2B4) at higher concentrations suggested that cyt b5 was occupying the cyt P450 reductase-binding site on cyt P450 2B4 and preventing the reduction of ferric cyt P450 (Zhang, H., Im, S.-C., and Waskell, L. (2007) J. Biol. Chem. 282, 29766-29776). In this work experiments were undertaken with manganese-containing cyt b5 (Mn-cyt b5) to test this hypothesis. Because Mn-cyt b5 does not undergo oxidation state changes under our experimental conditions, interpretation of the experimental results was unambiguous. The rate of electron transfer from cyt P450 reductase to ferric cyt P450 2B4 was decreased by Mn-cyt b5 in a concentration-dependent manner. Moreover, reduction of cyt P450 2B4 by cyt P450 reductase was incomplete in the presence of Mn-cyt b5. At a Mn-cyt b(5):cyt P450 2B4:cyt P450 reductase molar ratio of 5:1:1, the rate of reduction of ferric cyt P450 was decreased by 10-fold, and only 30% of the cyt P450 was reduced, whereas 70% remained oxidized. It could be demonstrated that Mn-cyt b5 had its effect by acting on cyt P450, not the reductase, because the reduction of cyt c by cyt P450 reductase in the presence of Mn-cyt b5 was not effected. Furthermore, under steady-state conditions in the cyt P450 reconstituted system, Mn-cyt b5, which lacks the ability to reduce oxyferrous cyt P450 2B4, was unable to stimulate the activity of cyt P450. Mn-cyt b5 only inhibited the cyt P450 2B4 activity. In conjunction with site-directed mutagenesis studies and experiments that strongly suggested that cyt b5 competed with cyt P450 reductase for binding to cyt P450, the current investigation demonstrates unequivocally that cyt b5 inhibits the activity of cyt P450 2B4 by preventing cyt P450 reductase from binding to cyt P450, a prerequisite for electron transfer from cyt P450 reductase to cyt P450 and catalysis.

Structural propensities in the heme binding region of apocytochrome b5. I. Free peptides

The water-soluble domain of rat microsomal cytochrome b5 is a small globular hemoprotein. Under native conditions, the apoprotein consists of a well-folded hydrophobic core and a 42-residue loop, which is substantially disordered in solution. Association with the heme cofactor causes the loop to organize into a second well-folded hydrophobic core encompassing four short helices, H2-H5. Of these, H3 and H4 are recognized as intrinsically disordered by algorithms that analyze primary structures for folding propensities. Three peptides, spanning H2-H5, H2-H3, and H4-H5, were designed, synthesized, and characterized to identify local structural preferences in the isolated loop. In addition, two replacements (D60R and N57P, which are known to stabilize holocytochrome b5) were introduced individually in the H4-H5 peptide. Helical content measured by nuclear magnetic resonance and far-UV circular dichroism spectroscopy in solutions of 2,2,2-trifluoroethanol revealed that H4 possessed a lower propensity to form the holoprotein structure than H3. Both replacements in H4 resulted in measurable changes in observed overall helical propensities. It was concluded that the prediction of intrinsic disorder was reliable. Furthermore, the stability of the holoprotein did not correlate simply with helical propensities in the disordered regions.

Optimalization of preparation of apocytochrome b5 utilizing apo-myoglobin

Cytochrome b5 (cyt b5), a component of endoplasmic reticulum membrane, plays a role in modulation of enzymatic activity of some cytochrome P450 (CYP) enzymes. The effect of apo-cytochrome b5 on this enzymatic system has not been investigated in details, because preparation of cyt b5 as a pure protein failed in many laboratories. In order to prepare the native apo-cytochrome b5 in a large scale we utilized a protein with higher affinity toward the heme; the apo-myoglobin from the equine skeletal muscle. In the first step, we extracted heme moiety from the native myoglobin by butanone extraction. Than the effect of pH on spontaneous heme release from both proteins was investigated: purified rabbit cyt b5 as well as equine skeletal muscle myoglobin. The prepared apo-myoglobin was incubated with the cyt b5 and heme transfer was monitored as a shift of absorption maximum from 413 to 409 nm in pH varying between 3–6 (10 mM KH2PO4, pH 3–6). Here, we obtained 43 mg of the equine skeletal muscle apo-myoglobin (43% yield). The optimal pH range for heme transfer from cyt b5 into apo-myoglobin was between 4.2 and 5. Native apo-cytochrome b5 was successfully prepared using procedure described here.

Forced Unfolding of Apocytochrome b 5 by Steered Molecular Dynamics Simulation

Apocytochrome b5 (apocyt b5), a small b-type cytochrome with heme prosthetic group removal, has been subjected to steered molecular dynamics (SMD) simulations for investigating the consequences of mechanical force-induced unfolding. Both constant velocity (0.5 and 1.0 A/ps) and constant force (500, 750 and 1000 pN) stretching have been employed to model forced unfolding of apocyt b5. The results of SMD simulations elucidate that apocyt b5 is protected against external stress mainly through the interstrand hydrogen bonding between its beta1-beta2 and beta2-beta3 strands, highlighting the importance of hydrophobic core 2 in stabilization of apocyt b5. The existence of intermediate states manifested by current simulations in the forced unfolding pathway of apocyt b5 is different from the observations in pervious thermal or chemical unfolding studies in the absence of force. The present study could thus provide insights into the relationship between the two cooperative functional modules of apocyt b5 and also guide the rational molecular design of heme proteins.

Retracted article: In-cell protein dynamics

The native intracellular environment of proteins is crowded with metabolites and macromolecules. However, most biophysical information concerning proteins is acquired in dilute solution. To determine whether there are differences in dynamics, nuclear magnetic resonance spectroscopy can be used to measure 15N relaxation in uniformly 15N-enriched apocytochrome b5 inside living Escherichia coli and in dilute solution. Such data can then be used to compare the fast backbone dynamics of the partially folded protein in cells to its dynamics in dilute solution by using Lipari-Szabo analysis. It appears that the intracellular environment does not alter the protein's structure, or significantly change its fast dynamics. Specifically, the cytosol does not change the amplitude of fast backbone motions, but does increase the average timescale of these motions, most likely due to the increase in viscosity of the cytosol.

A dynamic N‐capping motif in cytochrome b5: Evidence for a pH‐controlled conformational switch

Apocytochrome b5 is a marginally stable protein exhibiting under native conditions a slow conformational exchange in its C-terminal region. The affected elements of secondary structure include a 3(10)-helix containing at its N-terminus a histidine Ncap and a subsequent proline. Participation of the neutral histidine side-chain in backbone amide capping lowers the imidazole pKa. To explore the nature of the conformational exchange in the protein and determine whether it is related to cis-trans isomerization of the His-Pro bond, three octapeptides encompassing the helix were synthesized and studied by NMR spectroscopy. One corresponded to the wild-type sequence, the second was the D-histidine epimer, and the third contained an alanine in place of the proline. It was found that the rates of cis-trans interconversion in the proline-containing peptides were slower than the rates of the conformational exchange in the protein. In addition, the wild-type peptide hinted at a predisposition for Ncap formation when in the trans configuration. Analysis of the pH response of the peptides and protein suggested that at pH near neutral, the conformational exchange detected in the protein involved only species with a trans His-Pro bond and could be approximated with a three-state model by which the terminal helix sampled a locally unfolded state. This state, which contained an uncapped histidine with a normal pKa, partitioned into neutral and protonated populations according to pH. The intrinsic conformational bias of the wild-type peptide and the pH-driven equilibria illustrated how a 3(10)-element could serve as a nucleation site for structural rearrangement.

Protein Dynamics in Living Cells

A protein's structure is most often used to explain its function, but function also depends on dynamics. To date, protein dynamics have been studied only in vitro under dilute solution conditions where solute concentrations are typically less than 10 g/L, yet proteins function in a crowded environment where the solute concentration can exceed 400 g/L. Does the intracellular environment affect protein dynamics? The answer will help in assessing the biological significance of the NMR-derived dynamics data collected to date. We investigated fast protein dynamics inside living Escherichia coli by using in-cell NMR. The backbone dynamics of apocytochrome b5 were quantified using {1H}-15N nuclear Overhauser effect (nOe) measurements, which characterize motions on the pico- to nanosecond time scale. The overall trend of backbone dynamics remains the same in cells. Some of the nOe values differ, but most of the differences track the increased intracellular viscosity rather than a change in dynamics. Therefore, it appears that dilute solution steady-state {1H}-15N nOe measurements provide biologically relevant information about pico- to nanosecond backbone motion in proteins.

Structural and Dynamic Perturbations Induced by Heme Binding in Cytochrome b5,

The water-soluble domain of rat hepatic cytochrome b(5) undergoes marked structural changes upon heme removal. The solution structure of apocytochrome b(5) shows that the protein is partially folded in the absence of the heme group, exhibiting a stable module and a disordered heme-binding loop. The quality of the apoprotein structure in solution was improved with the use of heteronuclear NMR data. Backbone amide hydrogen exchange was studied to characterize cooperative units in the protein. It was found that this criterion distinguished the folded module from the heme-binding loop in the apoprotein, in contrast to the holoprotein. The osmolyte trimethylamine N-oxide (TMAO) did not affect the structure of the apoprotein in the disordered region. TMAO imparted a small stabilization consistent with an unfolded state effect correlating with the extent of buried surface area in the folded region of the native apoprotein. The failure of the osmolyte to cause large conformational shifts in the disordered loop supported the view that the specificity of the local sequence for the holoprotein fold was best developed with the stabilization of the native state through heme binding. To dissect the role of the heme prosthetic group in forcing the disordered region into the holoprotein conformation, the axial histidine belonging to the flexible loop (His63) was replaced with an alanine, and the structural properties of the protein with carbon-monoxide-ligated reduced iron were studied. The His63Ala substitution resulted in a protein with lower heme affinity but nevertheless capable of complete refolding. This indicated that the coordination bond was not necessary to establish the structural features of the holoprotein. In addition, the weak binding of the heme in this protein resulted in conformational shifts at a location distant from the binding site. The data suggested an uneven distribution of cooperative elements in the structure of the cytochrome.

The Kinetic and Spectral Characterization of the E. coli-Expressed Mammalian CYP4A7: Cytochrome b5 Effects Vary with Substrate

The CYP4A gene subfamily is composed of a number of genes that encode cytochromes P450 from various species, including human, which catalyze the hydroxylation of various saturated and unsaturated fatty acids, including arachidonic acid and prostaglandins. CYP4A7, a fatty acid metabolizing cytochrome P450 from rabbit kidney, was expressed in E. coli by adding the first 10 codons of CYP17α producing final yields of 20 nmol/L in order to perform detailed kinetic and spectral studies. CYP4A7 metabolized arachidonate, laurate, and myristate, with maximum turnover numbers of 152, 130, and 64.5 min−1 and corresponding Km values of 74.5, 27, and 16.7 μM, respectively, in the presence of cytochrome b5. In the absence of cytochrome b5, CYP4A7 metabolized laurate and myristate with turnover numbers of 27.4 and 33.6 min−1 and corresponding Km values of 3.9 and 33 μM, respectively. Arachidonate was not metabolized in the absence of cytochromeb5. Saturation kinetics studies performed with heme-depleted cytochrome b5 (apo cytochrome b5) yielded turnover numbers of 118 and 74 min−1 and Km values of 74 and 25 μM with laurate and myristate, respectively, indicating that cytochrome b5 is not involved in electron transfer but rather plays a conformational role. Laurate perturbation of the visible absorption spectrum of CYP4A7 allowed for determination of the spectral binding constant (KS) in the absence and presence of cytochrome b5 (13 and 43 μM, respectively). In stopped-flow kinetics experiments, the flavin reduction (∼90 s−1) and heme reduction (∼9 s−1) phases of the monooxygenase reaction of CYP4A7 were not altered by the presence of cytochrome b5. Estimations of the rate of CPR (0.3 s−1) or cytochrome b5 (9.1 s−1) binding with CYP4A7 were also determined.

Defining folding and unfolding reactions of apocytochrome b5 using equilibrium and kinetic fluorescence measurements.

The refolding and unfolding kinetics of a soluble domain of apocytochrome b5 extending from residue 1 to 104 have been characterized using stopped flow and equilibrium-based fluorescence methods. The isolated apoprotein unfolds reversibly in the presence of GuHCl. From cooperative unfolding curves, the conformational stability (Delta G(uw)), in the absence of denaturant, is estimated to be 11.6 +/- 1.5 kJ mol-1 at 10 degrees C. The stability of apocytochrome b5 is lower than that of the corresponding form of the holoprotein (Delta G approximately 25 kJ mol-1) and exhibits a transition midpoint at 1.6 M GuHCl. Kinetic studies support the concept of a two-state model with both unfolding and refolding rates showing an exponential dependence on denaturant concentration with no evidence of the formation of transient intermediates in either limb of the chevron plot. Apocytochrome b5 is therefore an example of a protein in which both kinetics and equilibria associated with folding are described by a two-state model. The values of mku and mkf obtained from kinetic analysis are an indication of a transition state (mku/meq of 0.29) that resembles the native form by retaining similar solvent accessibility and many of the noncovalent interactions found in the apoprotein. The changes in heat capacity support a transition state that resembles the apoprotein with a value for Delta Cpf of -3.6 kJ mol-1 K-1 estimated for the refolding reaction. From these measurements, a model of refolding that involves the rapid nucleation of hydrophobic residues around Trp26 is suggested as a major event in the formation of the native apoprotein.

Backbone Dynamics of Apocytochrome b(5) in Its Native, Partially Folded State.

The backbone dynamics in the native state of apocytochrome b5 were studied using 15N nuclear magnetic spin relaxation measurements. The field (11.7 and 14.1 T) and temperature (10-25 degrees C) dependence of the relaxation parameters (R1, R2, and R1rho) and the 1H-15N NOE established that the protein undergoes multiple time scale internal motions related to the secondary structure. The relaxation data were analyzed with the reduced spectral density mapping approach and within the extended model-free framework. The apoprotein was confirmed to contain a disordered heme-binding loop of approximately 30 residues with dynamics on the sub-nanosecond time scale (0.6 0.75, taue 300 micros) than for the folded polypeptide chain (tauex < 150 micros). The distribution of the reduced spectral densities at the 1H and 15N frequencies followed the dynamic trend and predicted the slowing of the internal motions at 10 degrees C. Comparison of the dynamics with those of the holoprotein [Dangi, B., Sarma, S., Yan, C., Banville, D. L., and Guiles, R. D. (1998) Biochemistry 37, 8289-8302] demonstrated that binding of the heme alters the time scale of motions both in the heme-binding loop and in the structured hydrophobic core.

Backbone dynamics of apocytochrome b5 in its native, partially folded state.

The backbone dynamics in the native state of apocytochrome b5 were studied using 15N nuclear magnetic spin relaxation measurements. The field (11.7 and 14.1 T) and temperature (10-25 degrees C) dependence of the relaxation parameters (R1, R2, and R1rho) and the 1H-15N NOE established that the protein undergoes multiple time scale internal motions related to the secondary structure. The relaxation data were analyzed with the reduced spectral density mapping approach and within the extended model-free framework. The apoprotein was confirmed to contain a disordered heme-binding loop of approximately 30 residues with dynamics on the sub-nanosecond time scale (0.6 < S2 < 0.7, 100 ps < taue < 500 ps). This loop is attached to a structured hydrophobic core, rigid on the picosecond time scale (S2 > 0.75, taue < 50 ps). The inability to fit the data for several residues with the model-free protocol revealed the presence of correlated motion. An exchange contribution was detected in the transverse relaxation rate (R2) of all residues. The differential temperature response of R2 along the backbone supported slower exchange rates for residues in the loop (tauex > 300 micros) than for the folded polypeptide chain (tauex < 150 micros). The distribution of the reduced spectral densities at the 1H and 15N frequencies followed the dynamic trend and predicted the slowing of the internal motions at 10 degrees C. Comparison of the dynamics with those of the holoprotein [Dangi, B., Sarma, S., Yan, C., Banville, D. L., and Guiles, R. D. (1998) Biochemistry 37, 8289-8302] demonstrated that binding of the heme alters the time scale of motions both in the heme-binding loop and in the structured hydrophobic core.

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.

Structural consequences of heme removal: molecular dynamics simulations of rat and bovine apocytochrome b5.

Molecular dynamics simulations of rat and bovine apocytochrome b5 were performed to investigate the structural and dynamical consequences of heme removal. A crystal structure is available for the bovine holoprotein, while experimental studies of apocytochrome b5 have focused on the rat protein. The rat and bovine proteins are 93% homologous by sequence, and the sequence differences (six residues) appear to have no effect on the structure of the native holoprotein, as seen by the correlation of a bovine simulation with rat holocytochrome b5 experimental data (Storch & Daggett, 1995). There was a marked effect, however, on the structure and dynamics of the apo form. The bovine apocytochrome b5 simulation displayed subtle inconsistencies when compared to the experimental results on the rat apoprotein. Therefore, the rat protein was constructed from the bovine crystal structure coordinates. The MD simulation of the rat apoprotein displayed greater deviations from the crystal structure, yet it was in much closer agreement to the experimental data for the apoprotein. Additionally, the six variant residues fall in the regions where the bovine protein deviated from experiment. The two hydrophobic cores of the rat protein behaved very differently. Core 2 was well maintained, retained native-like structure, and is in good agreement with NMR data (Moore & Lecomte, 1990). Conversely, core 1, which is normally constrained by the prosthetic heme group, exhibited conformational heterogeneity, increased mobility, and some loss of secondary structure. Thus, the model of rat apocytochrome b5 complements past studies by providing structural information about core 1 that has proved difficult to obtain by experiment. The bovine simulation serves as a prediction, since little to no experimental data exist for this form of the apoprotein.