Ferricytochrome B5 Research Articles

Evaluation of the Cytochrome b5/Cytochrome b5 Reductase Pathway

AbstractNADH cytochrome b5 reductase (b5R; EC 1.6.2.2; Diaphorase I; NADH: ferricytochrome b5 oxidoreductase) is an FAD‐containing protein, which, along with the hemoprotein cytochrome b5 (cyt b5), mediates electron transfer from NADH to fatty acid desaturases, P450 oxidases, methemoglobin, and ascorbyl free radical. In addition, b5R and cyt b5 can directly catalyze the reduction of hydroxylamine and amidoxime metabolites. This unit provides protocols for measuring the activity and mRNA expression of the cytochrome b5/cytochrome b5 reductase pathway, and for obtaining heterologous expression and purification of the soluble forms of each protein.

The orientations of cytochrome c in the highly dynamic complex with cytochrome b5 visualized by NMR and docking using HADDOCK.

The interaction of bovine microsomal ferricytochrome b5 with yeast iso-1-ferri and ferrocytochrome c has been investigated using heteronuclear NMR techniques. Chemical-shift perturbations for 1H and 15N nuclei of both cytochromes, arising from the interactions with the unlabeled partner proteins, were used for mapping the interacting surfaces on both proteins. The similarity of the binding shifts observed for oxidized and reduced cytochrome c indicates that the complex formation is not influenced by the oxidation state of the cytochrome c. Protein-protein docking simulations have been performed for the binary cytochrome b5-cytochrome c and ternary (cytochrome b5)-(cytochrome c)2 complexes using a novel HADDOCK approach. The docking procedure, which makes use of the experimental data to drive the docking, identified a range of orientations assumed by the proteins in the complex. It is demonstrated that cytochrome c uses a confined surface patch for interaction with a much more extensive surface area of cytochrome b5. Taken together, the experimental data suggest the presence of a dynamic ensemble of conformations assumed by the proteins in the complex.

Interprotein electron transfer in a confined space: uncoupling protein dynamics from electron transfer by sol-gel encapsulation.

In this paper, we describe the first observations of photoinitiated interprotein electron transfer (ET) within sol-gels. We have encapsulated three protein-protein complexes, specifically selected because they represent a full range of affinities, are sensitive to different types of dynamic processes, and thus are expected to respond differently to sol-gel encapsulation. The three systems are (i) the [Zn, Fe(3+)L] mixed-metal hemoglobin hybrids, where the alpha(1)-Zn and beta(2)-Fe subunits correspond to a "predocked" protein-protein complex with a crystallographically defined interface (Natan, M. J.; Baxter, W. W.; Kuila, D.; Gingrich, D. J.; Martin, G. S.; Hoffman, B. M. Adv. Chem. Ser. 1991, 228 (Electron-Transfer Inorg., Org., Biol. Syst.), 201-213), (ii) the Zn-cytochrome c peroxidase complex with cytochrome c, [ZnCcP, Fe(3+)Cc], having an intermediate affinity between its partners (Nocek, J. M.; Zhou, J. S.; De Forest, S.; Priyadarshy, S.; Beratan, D. N.; Onuchic, J. N.; Hoffman, B. M. Chem. Rev. 1996, 96, 2459-2489), and (iii) the [Zn-deuteromyoglobin, ferricytochrome b(5)] complex, [ZnDMb, Fe(3+)b(5)], which is loosely bound and highly dynamic (Liang, Z.-X.; Nocek, J.; Huang, K.; Hayes, R. T.; Kurnikov, I. V.; Beratan, D. N.; Hoffman, B. M. J. Am. Chem. Soc. 2002, 124, 6849-6859. Intersubunit ET within the hybrid does not involve second-order processes or subunit rearrangements, and thus is influenced only by perturbations of high-frequency motions coupled to ET. For the latter two complexes, sol-gel encapsulation eliminates second-order processes: protein partners encapsulated as a complex must stay together throughout a photoinitiated ET cycle, while proteins encapsulated alone cannot acquire a partner. It further modulates intracomplex motions of the two partners.

Influence of site-directed mutation of cytochrome b5 Phe35 on the protein’s structure and properties

Kinetic studies of the heme dissociation from the wild type and Phe35Tyr, Phe35Leu mutants of bovine liver microsomal ferricytochrome b5 indicate that the oxidized Phe35Tyr mutant is more stable towards denaturant than wild type but Phe35Leu mutant proceeded with a different mechanism compared with wild type cytochrome b5 and Phe35Tyr mutant protein. Because of the decrease of side chain volume in Phe35Leu mutant, a cavity produced in the interior of the protein may offer a channel for urea molecule to enter the hydrophobic pocket. When urea concentration is larger than 5 mol/L, the urea molecule may compete to coordinate the iron of heme with His39, that results in sharp increase of the rate of heme dissociation. The interaction between cytochrome b5 and cytochrom c demonstrated that a 1:1 protein complex was formed between the two proteins. The binding constants of cytochrome b5 with cytochrome c are: wild typeK A = 4.2(±0.01) × 106(mol/L)−1, Phe35TyrK A = 3.7(±0.01) × 106 (mol/L)−1 and Phe35LeuK A = 4.7(±0.01) × 106(mol/L)−1 respectively (I = l m mol/L, pH 7.0 soldium phosphate buffer, 25°C). These results clearly show that the mutation at Phe35 has no influence on the binding of cytochrome b5 with cytochrome c and that the hydrophilic patch residues are not involved in the binding of cytochrome b5 and cytochrome c.

An1H–13C–13C-Edited1H NMR Experiment for Making Resonance Assignments in the Active Site of Heme Proteins

In paramagnetic heme proteins, it is often problematic to make proton resonance assignments for heme substituents that do not have large isotropic shifts and consequently lie under the large envelope of polypeptide resonances. Furthermore, assignments that would normally be performed with the aid of HMBC experiments in diamagnetic molecules can prove difficult in the active site of paramagnetic heme proteins ifT−12>2JCH. To circumvent this problem, a new method is presented to selectively detect1H in1Hn–13C–13C fragments biosynthetically introduced into the active site of heme proteins. The pulse sequence combines well-known building blocks such as INEPT to transfer1H spin magnetization to bonded13C nuclei, followed by INADEQUATE to generate13C–13C double-quantum coherence that is selected with pulsed field gradients, and finally reverse-INEPT to transfer magnetization back to1H nuclei for subsequent observation. The new1Hn–13C–13C edited experiment takes advantage of the relatively large values of1JCHand1JCC, avoiding the long interpulse delays in HMBC that compromise the detectability of rapidly relaxing nuclei. The potential applicability of the pulse sequence is demonstrated by its contribution to the unambiguous assignment of the carbonyl carbons in the heme propionates of ferricytochrome b5.

Analysis of backbone dynamics in cytochrome b5 using 15N-NMR relaxation measurements.

Determination of 15N-NMR relaxation rates has allowed the characterisation of the dynamic properties of 55 1H-15N bond vectors in a soluble haem-binding domain of bovine microsomal ferricytochrome b5 consisting of the first 104 amino acid residues. Measurements of heteronuclear [1H]-15N-NMR nuclear Overhauser effects, and 15N-NMR longitudinal and transverse relaxation rates have been analysed using both model-free and reduced spectral density mapping approaches, demonstrating the application of these methods to a paramagnetic system. Analysis reveals that, for those regions which have been assessed, the polypeptide backbone of ferricytochrome b5 is largely rigid (average S2 = 0.80), and that minimal internal backbone motion occurs on the sub-nanosecond timescale. In contrast, motions on the microsecond to millisecond timescale, especially pronounced for the loop region extending from residues 28 to 31, and which may be of biological relevance, are indicated.

Analysis of the paramagnetic shifts of haem carbon resonances in bovine ferricytochrome b5.

Recently published chemical shifts for haem 13C nuclei in bovine ferricytochrome b5 (Lee KB, Kweon J, Park H (1995) Assignment of hyperfine-shifted heme carbon resonances in ferricytochrome b5. FEBS Lett. 367:77-80) are analysed in terms of haem molecular orbitals with perturbed D4h symmetry. Since a crystal structure of this protein is available, together with extensive 1H assignments both in the oxidised and reduced forms, the paramagnetic shifts can be separated into dipolar and Fermi contact contributions by using an empirical magnetic susceptibility tensor. The results are compared with the orientation of the tensor and the geometry of the haem ligands. This comparison casts doubt on one of the 13C assignments and demonstrates that the asymmetry of the haem electronic structure is dominated by the influence of both of the His ligands. The 13C chemical shifts of two haem methyl groups in the minor form of the protein, in which the haem is approximately rotated by 180 degrees about its 5CH-15CH axis, are also evaluated.

13C NMR spectroscopic and X-ray crystallographic study of the role played by mitochondrial cytochrome b5 heme propionates in the electrostatic binding to cytochrome c.

The role played by the outer mitochondrial membrane (OM) cytochrome b5 heme propionate groups in the electrostatic binding between OM cytochrome b5 and horse heart cytochrome c was investigated by 13C NMR spectroscopy and X-ray crystallography. To achieve these aims, 13C-labeled heme OM cytochrome b5 was expressed in Escherichia coli as previously described [Rivera M., Walker, F.A. (1995) Anal. Biochem. 230, 295-302]. Assignment of the resonances arising from the heme propionate carbons in ferricytochrome b5 was carried out by a combination of one- and two-dimensional NMR experiments. Titrations of [13C]heme-labeled OM cytochrome b5 with horse heart cytochrome c were carried out in order to monitor the resonances arising from the heme propionate carbonyl carbons in OM cytochrome b5. The results from these titrations clearly show that only the heme propionate located on the exposed heme edge in OM cytochrome b5 participates in the electrostatic stabilization of the complex between OM cytochrome b5 and horse heart cytochrome c. Similar experiments carried out monitoring 13C resonances arising from several other heme substituents demonstrated that the stoichiometry of the complex is 1:1. A conditional binding constant, K which equals 3.8 x 10(4) +/- 1.4 x 10(4) at mu = 0.02 M, was obtained for the formation of the complex by fitting the binding curves obtained experimentally to a model based on this stoichiometry. The X-ray crystal structure of rat liver OM cytochrome b5 solved to 2.7 A resolution shows that the structures of bovine liver microsomal cytochrome b5 and rat liver OM cytochrome b5 are almost identical when compared at medium resolution. The similarity between the two structures, combined with the findings that only the heme propionate located on the exposed heme edge of OM cytochrome b5 participates in the electrostatic binding to cytochrome c and that the stability of this complex is similar to that measured for the association between microsomal cytochrome b5 and cytochrome c, clearly indicates that the site of interaction on OM cytochrome b5 is almost identical to the one elucidated for microsomal cytochrome b5. It is therefore possible to conclude that the large body of information gathered by many investigators for the nonphysiological interaction between microsomal cytochrome b5 and cytochrome c (recently reviewed) [Mauk, A. G. Mauk, M. R., Moore, G. R., & Northrup, S. H. (1995) Bioenerg. Biomembr. 27, 311-330] has indeed biological as well as pedagogical validity.

Electrochemical measurement of second-order electron transfer rate constants for the reaction between cytochrome b5 and cytochrome c.

The second-order electron transfer reaction between cytochrome b5 and cytochrome c has been studied by cyclic voltammetry utilizing a gold electrode modified with beta-mercaptopropionate. When cyclic voltammetry is performed on a solution containing a mixture of cytochrome b5, cytochrome c and polylysine, cytochrome b5 undergoes reversible electrochemistry at the electrode surface while cytochrome c discriminates against the electrode surface. The selectivity of the modified electrode for negatively charged proteins makes it possible to selectively reduce a protein possessing a net negative charge and a relatively low reduction potential (outer mitochondrial membrane cytochrome b5, Eo = -102 mV; microsomal cytochrome b5, Eo = 3 mV) in the presence of another protein possessing a net positive charge and a relatively high reduction potential (cytochrome c, Eo = 265 mV). The electrochemical reduction of ferricytochrome b5 at the electrode surface is followed by a second-order electron transfer reaction between ferrocytochrome b5 and ferricytochrome c that yields ferricytochrome b5 and ferrocytochrome c. This fast homogeneous electron transfer reaction which is preceded by a heterogeneous electron transfer reaction results in a characteristic cyclic voltammogram containing a pre-peak to the reduction current. The second-order rate constant for the homogeneous reaction was obtained by invoking the above reaction scheme for digital simulation of a cyclic voltammogram which was subsequently fitted to the experimental data. Second-order rate constants obtained with this method are 2.9 x 10(8) and 8.9 x 10(8) for the homogeneous electron transfer reactions between rat liver outer mitochondrial membrane (OM) ferrocytochrome b5 and beef liver microsomal ferrocytochrome b5, with horse heart ferricytochrome c, respectively. These values are in good agreement with second-order rate constants obtained for the same protein systems by flash photolysis. [Meyer, T. E., Rivera, M., Walker, F. A., Mauk, M. R., Mauk, A. G., Cusanovich, M. A., & Tollin, G. (1993) Biochemistry 32, 622-627].

Assignment of hyperfine-shifted heme carbon resonances in ferricytochrome b5

The reverse detection heteronuclear multiple quantum coherence, HMQC, study of native bovine ferricytochrome b 5 has provided the complete assignment of hyperfine shifted resonances of heme carbons attached proton(s). The dominant delocalized π-spin density to vinyl groups gives rise to contact shifts which have opposite direction for a carbon and its attached proton(s). The most hyperfine shifted 13C heme signals are mainly generated from 3rd heme pyrrole ring substituents which identifies that the molecular orbital for facile electron transfer is oriented to exposed heme edge. Magnetic/electronic asymmetry of heme induced by two axial His makes spread the hyperfine shifted heme carbon resonances over the range of 280 ppm at 25°C, which would be the more sensitive probe than those of proton resonances in characterizing the nature of heme electronic structure of ferricytochrome b 5.

Photoinduced electron transfer from the triplet state of zinc cytochrome c to ferricytochrome b5 is gated by configurational fluctuations of the diprotein complex.

The intracomplex electron-transfer reaction 3Zncyt/b5(III)-->Zncyt+/b5(II) within electrostatic and covalent complexes of zinc(II) cytochrome c and ferricytochrome b5 is studied by laser flash photolysis. Kinetic effects of protein cross-linking and of solution viscosity are interpreted in terms of dynamic mobility of the associated proteins with respect to each other. The rate constant for the monoexponential reaction in the electrostatic complex is 3.5 x 10(5) s-1 in aqueous solution; this value is independent of protein concentration and ionic strength, but it decreases markedly as viscosity is raised by addition of glycerol or sucrose. The multiexponential reaction in the covalent complex was analyzed also in terms of the stretched exponential, exp[-(kt)n]. The best fit requires k = 6.8 x 10(4) s-1 and n = 0.56 in aqueous solution; this rate constant is independent of protein concentration and ionic strength, but it decreases slightly as viscosity is raised. Fitting of the viscosity dependence to a simple two-state kinetic model yields a rate constant of 3.0 x 10(5) s-1 for rearrangement of the electrostatic complex 3Zncyt/b5(III) from the initial docking configuration to a different, more reactive, configuration. The corresponding rate constant for rearrangement of the electrostatic complex 3Zncyt/pc(II) containing plastocyanin, determined previously, is 2.5 x 10(5) s-1. Evidently, the intracomplex reaction in both electrostatic complexes is gated by a rearrangement process.(ABSTRACT TRUNCATED AT 250 WORDS)

Hydrogen isotope effects on the proton nuclear magnetic resonance spectrum of bovine ferricytochrome b5: Axial hydrogen bonding involving the axial His-39 imidazole ligand

The potential role of hydrogen bonding interactions in modulating the molecular and electronic structure of the active site of solubilized bovine ferricytochrome b 5 has been investigated by monitoring solvent isotope effects on proton-NMR spectral parameters. It is observed that the hyperffine shifts of both the heme prosthetic group and one coordinated His are sensitive, while those for the other axial His and non-coordinated residues are insensitive, to 2H for 1H exchange. Two types of isotope influences are characterized; one whose chemical shift influence is time-resolved on the NMR rime scale, and involves a single proton on one axial ligand, and a second effect which involves multiple protons, is not time resolved, and influences primarily the heme. A large isotope effect on the hyperfine shift is identified for the C β H signals of His-39 but not His-63. The exchangeable ring NH of His-39 is assigned, and the pH influence on the exchange properties of heme pocket labile protons, when compared to the rate of base catalyzed averaging of the His-39 C β H isotope effect, lead to the conclusion that the axial hydrogen bond which is responsible for this isotope effect is that between His-39 ring NH and Gly-42 carbonyl. The more rapid exchange of labile protons with solvent for His-63 than His-39 confirms a less solvent accessible and stronger hydrogen bonded His-39 than His-63. The stronger His-39-Gly-42 than His-63-Phe-58 hydrogen bond involving the ring NH leads to more extensive His-39 imidazolate character and hence a stronger iron-His-39 than iron-His-63 bond. The much larger hyperfine shifts for His-39 than His-63 imidazole ring non-labile protons support the stronger bonding of the former ligand, and account for the orientation of the rhombic magnetic axes by His-39 rather than His-63. The solvent isotope effect on the heme leads to rotation of the prosthetic group about the His-Fe-His bond by ≈ 0.5° so as to shorten the 7-propionate link to Ser-64. This suggests that the hydrogen bonds between the 7-propionate group and Ser-64 are responsible for the effect.

The expression of bovine microsomal cytochrome b5 in Escherichia coli and a study of the solution structure and stability of variant proteins.

The DNA sequence of bovine microsomal cytochrome b5 has been amplified from a liver cDNA library using a polymerase chain reaction. The amplified cDNA when cloned into plasmids that support the high-level production of cytochrome b5 in E.coli leads to protein overexpression and results in cell colonies bearing a strong red colouration. Using cassette mutagenesis, truncated versions of the cytochrome b5 cDNA have been made that encode the first 90 amino acid residues (Ala1-Lys90), the first 104 amino acids (Ala1-Ser104) and the complete protein (Ala1-Asn133). The location of the overexpressed cytochrome b5 within prokaryotic cells is dependent on the overall length of the protein. Expression of the Ala-Lys90 and Ala1-Ser104 variants leads to a location in the cytoplasmic phase of the bacteria whereas the whole protein, Ala1-Asn133, is found within the bacterial membrane fraction. The last 30 residues of cytochrome b5 therefore contain all of the necessary information to insert the protein into E.coli membranes. The solubility of the Ala1-Ser104 variant permits the solution structure and stability of this protein to be measured using 1- and 2-D 1H-NMR methods and electronic spectroscopy. 1-D NMR studies show that the chemical shifts of the haem and haem ligand resonances of the Ala1-Ser104 variant exhibit only very slight perturbations to their magnetic microenvironments when compared with the tryptic fragment of ferricytochrome b5. These results indicate an arrangement of residues in the haem pocket that is very similar in both the Ala1-Ser104 variant and the tryptic fragment and by 2-D NMR it is shown that this similarity extends to the conformations of the polypeptide backbone and side chains. Electronic spectroscopy of this variant shows absorbance maxima for the Soret peaks at 423 nm (reduced) and 413 nm (oxidized). From absorbance spectra the relative thermal stabilities of the Ala1-Ser104 variant and the tryptic fragment were measured. In the oxidized state the Ala1-Ser104 variant denatures in a single cooperative transition with a midpoint temperature (Tm) of 73 degrees C that is significantly higher than that of 'tryptic' ferricytochrome b5. The reduced form of the protein shows increased transition temperatures (Tm approximately 78 degrees C) reflected in the values of delta Hm, delta Sm and delta(delta G) of 420 kJ/mol, 1096 J/mol/K and 12.38 kJ/mol respectively, estimated for this variant. The increased stability of the Ala1-Ser104 variant and other recombinant forms of cytochrome b5 is correlated with the presence of additional residues at the N- and C-termini.(ABSTRACT TRUNCATED AT 400 WORDS)

The identification of cation‐binding domains on the surface of microsomal cytochrome b5 using 1H‐NMR paramagnetic difference spectroscopy

One-dimensional and two-dimensional 1H-NMR methods and paramagnetic difference spectroscopy have defined cation binding domains on the surface of the tryptic fragment of microsomal cytochrome b5. The addition of tris(ethylenediamine) chromium(III) [Cr(en)3(3+)] to solutions of ferricytochrome b5 reveals at least three distinct sites on the surface of the protein to which highly charged cations may bind (20 mM phosphate pH 7.0, T = 300 K). Surprisingly only one of these sites is located close to the haem edge region of the protein, whilst the remaining two sites are more remote. Site I contains the exposed haem C13 propionate and a series of carboxylate residues that includes glutamates 37, 38, 43, 44, and 48. Sites II and III are located away from the haem edge region and are delineated by the broadening of aromatic resonances of histidines 26 and 80. Further investigation of the interaction between Cr(en)3(3+) and cytochrome b5 using two-dimensional double-quantum-filtered correlated spectroscopy shows that resonances assigned to Glu59, Asp60, Glu79, Asp82 and Asp83 are broadened with the distribution of these charged side chains correlating with the relaxation broadening observed from one-dimensional experiments. In a binary complex with ferricytochrome c, Cr(en3(3+) broadens many cytochrome b45 resonances including the haem propionates, His26, Ala54, Thr55 and His80. Although the pattern of line-broadening of resonances at sites II and III is unaltered by complex formation, cytochrome c shields residues at site I, the haem edge site. The results indicate that the interaction between cytochrome b5 and c in a binary complex involves multiple protein configurations.

Electron transfer between horse ferritin and ferrihaemoproteins.

Reactions of reduced horse spleen ferritin with horse and Saccharomyces cerevisiae ferricytochromes c, cow ferricytochrome b5, sperm-whale metmyoglobin and Pseudomonas aeruginosa ferricytochrome c-551 were investigated by u.v.-visible spectrophotometry. In all cases the reduced ferritin reduced the ferrihaemoproteins. The rate of reduction varied from less than 0.2 M-1.s-1 for metmyoglobin to 1.1 x 10(3) M-1.s-1 for horse ferricytochrome c (0.1 M-phosphate buffer, pH 7.4, at 25 degrees C). We conclude that the mechanism of ferrihaemoprotein reduction involves long-range electron transfer through the coat of ferritin and that such electron transfer is rapid enough to account for the rates of iron release observed by other workers in reductive release assays.

Proton NMR study of the influence of hydrophobic contacts on protein prosthetic group recognition in bovine and rat ferricytochrome b5

The proton nuclear magnetic resonance spectra of the soluble fragment of native bovine and genetically engineered wild-type rat ferricytochrome b5 reconstituted with a wide variety of hemes chemically modified at 2- and/or 4-positions have been recorded and analyzed. While all but one nonsymmetric heme yielded comparable amounts of the two heme orientations immediately after reconstitution, the relative proportion of the two orientations at equilibrium varied widely. The unpaired spin density distribution in the heme pi system leads to substituent hyperfine shift patterns in these paramagnetic complexes that are completely diagnostic of the heme orientation in the protein matrix. An empirical assignment strategy is outlined and applied which allows unequivocal assignment of the absolute orientation of a derivatized heme within the protein matrix. Using a series of hemes lacking 2-fold symmetry solely due to a single substitution, the preferences for localized site occupation of vinyls, methyls, and hydrogens are developed. The large differences in relative stability of the two orientations of native protohemin in the two cytochromes b5 is shown to result from the additivity of localized effects for the bovine protein and the near cancellation of competing effects in the rat protein. The major determinant of the heme orientation is judged to be a repulsive interaction between a vinyl and a hydrophobic cluster of amino acids including positions 23 and 25. The differences in this heme orientational preference among bovine, rat, and chicken ferricytochromes b5 could be correlated with the relative steric bulk of the residues at positions 23 and 25.(ABSTRACT TRUNCATED AT 250 WORDS)

The formation of protein complexes between ferricytochrome b5 and ferricytochrome c studied using high‐resolution 1H‐NMR spectroscopy

The association of the tryptic fragment of bovine microsomal cytochrome b5 with cytochrome c has been studied by one- and two-dimensional 1H-NMR spectroscopy. The association of cytochromes to form protein complexes is apparent from the increase in linewidths for resonances of ferricytochrome b5 as well as small perturbations in their chemical shifts that occur upon increasing the cytochrome c/b5 molar ratio. The changes in the chemical shifts of hyperfine shifted resonances of ferricytochrome b5 with increasing ratios of ferricytochrome c indicate the formation of binary 1:1 complexes and ternary 1:2 complexes. Similarly, titrations of the linewidth of resolved resonances of ferricytochrome b5 are consistent with stoichiometries of 1:1 and 1:2 for complexes formed between cytochromes b5 and c. Surprisingly, in the 1:1 complex, mobility is shown to be a function of ionic strength. Two-dimensional correlated spectroscopy (COSY) and nuclear Overhauser enhancement spectroscopy (NOESY) of the binary complex formed between ferricytochrome b5 and c indicate that the positions of many resonances attributable to amino acids are unaltered by protein association, although distinctive chemical shift changes are detected in the alpha-CH of the haem C17 propionate. The protein complex detected by NMR is discussed with respect to the model for the binary complex proposed by Salemme and possible mechanisms of electron transfer.

One- and two-dimensional nuclear Overhauser effect studies of the eletronic/molecular structure of the heme cavity of ferricytochrome b5

A nuclear Overhauser effect, NOE, study of solubilized native bovine ferricytochrome b 5 has provided the complete assignment of the heme resonances as well as those of the majority of the amino acid side-chains making contact with the prosthetic group. The resonances which could not be identified are those from positions very close to the iron (< 5Å) for which paramagnetic relaxation is sufficiently strong to significantly decrease the NOEs. The observed 1H- 1H dipolar contacts generally confirm a solution structure unchanged from that described in single crystals, except for the detailed orientation of the heme side-chains. The 2-vinyl group is found in both the cis and trans in-plane orientation as opposed to exclusively cis in the crystal, and the 7-propionate group is rotated by 30° in solution towards the 6-propionate group. Identification of resonances for the individual axial histidine residues indicates non-equivalent interaction with the heme iron, and the patterns of meso-H, pyrrole substituent and amino acid dipolar shifts allow the location of the principal magnetic axes in the protein coordinate system. This identifies His-39 as the dominant influence in determining the electronic ground state that orients the molecular orbital for facile electron transfer via the exposed heme edge. The complete two-dimensional NOESY map for ferricytochrome b 5 is presented that yields all the cross peaks expected on the basis of the one-dimensional NOE studies, and indicates that such two-dimensional methods should have profitable extension to strongly hyperfine-shifted resonances in paramagnetic proteins.

ChemInform Abstract: Ferricytochrome b5: Assignment of Heme Propionate Resonances on the Basis of Nuclear Overhauser Effect Measurements and the Nature of Interprotein Contacts with Partner Redox Proteins.

Chemischer InformationsdienstVolume 17, Issue 28 Physical Organic Chemistry ChemInform Abstract: Ferricytochrome b5: Assignment of Heme Propionate Resonances on the Basis of Nuclear Overhauser Effect Measurements and the Nature of Interprotein Contacts with Partner Redox Proteins. S. J. MCLACHLAN, S. J. MCLACHLANSearch for more papers by this authorG. N. LA MAR, G. N. LA MARSearch for more papers by this authorE. SLETTEN, E. SLETTENSearch for more papers by this author S. J. MCLACHLAN, S. J. MCLACHLANSearch for more papers by this authorG. N. LA MAR, G. N. LA MARSearch for more papers by this authorE. SLETTEN, E. SLETTENSearch for more papers by this author First published: July 15, 1986 https://doi.org/10.1002/chin.198628068AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article. Volume17, Issue28July 15, 1986 RelatedInformation

Ferricytochrome b5: assignment of heme propionate resonances on the basis of nuclear Overhauser effect measurements and the nature of interprotein contacts with partner redox proteins

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTFerricytochrome b5: assignment of heme propionate resonances on the basis of nuclear Overhauser effect measurements and the nature of interprotein contacts with partner redox proteinsStuart J. McLachlan, Gerd N. La Mar, and Einar. SlettenCite this: J. Am. Chem. Soc. 1986, 108, 6, 1285–1291Publication Date (Print):March 1, 1986Publication History Published online1 May 2002Published inissue 1 March 1986https://doi.org/10.1021/ja00266a026RIGHTS & PERMISSIONSArticle Views82Altmetric-Citations36LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (814 KB) Get e-Alerts Get e-Alerts