Bovine Microsomal Cytochrome B5 Research Articles

Solution structure of cytochrome b5 mutant (E44/48/56A/D60A) and its interaction with cytochrome c

Using 1617 meaningful NOEs with 188 pseudocontact shifts, a family of 35 conformers of oxidized bovine microsomal cytochrome b5 mutant (E44/48/56A/D60A) has been obtained and is characterized by good resolution (rmsd to the mean structure are 0.047 +/- 0.007 nm and 0.095 +/- 0.008 nm for backbone and heavy atoms, respectively). The solution structure of the mutant, when compared with the X-ray structure of wild-type cytochrome b(5), has no significant changes in the whole folding and secondary structure. The binding between cytochrome b(5) and cytochrome c shows that the association constant of the mutant-cytochrome c complex is much lower than the one for wild-type complex (2.2 x 10(4) M(-1) vs. 5.1 x 10(3) M(-1)). The result suggests the four acidic residues have substantial effects on the formation of the complex between cytochrome b(5) and cytochrome c, and therefore it is concluded reasonably that the electrostatic interaction plays an important role in maintaining the stability and specificity of the complex formed. The competition between the ferricytochrome b(5) mutant and [Cr(oxalate)(3)](3-) for ferricytochrome c shows that site III of cytochrome c, which is a strong binding site to wild-type cytochrome b(5), still binds to the mutant with relatively weaker strength. Our results indicate that certain bonding geometries do occur in the interaction between the present mutant and cytochrome c and these geometries, which should be quite different from the ones of the Salemme and Northrup models.

Analysis of folding and unfolding reactions of cytochrome b5.

The guanidine hydrochloride- (GuHCl-) induced unfolding and refolding of a recombinant domain of bovine microsomal cytochrome b(5) containing the first 104 amino acid residues has been characterized by both transient and equilibrium spectrophotometric methods. The soluble domain is reversibly unfolded and the equilibrium reaction may be monitored by changes in absorbance and fluorescence that accompany denaturation of the native protein. Both probes reveal a single cooperative transition with a midpoint at 3 M GuHCl and lead to a value for the protein stability (DeltaG(uw)) of 26.5 kJ mol(-1). This stability is much higher than that reported for the corresponding form of the apoprotein (approximately 7 kJ mol(-1)). Transient changes in fluorescence and absorbance during protein unfolding exhibit biphasic profiles. A fast phase occupying approximately 30% of the total amplitude is observed at high denaturant concentrations and becomes the dominant process within the transition region. The rates associated with each process show a linear dependency on GuHCl concentration, and at zero denaturant concentration the unfolding rates (k(uw)) are 4.5 x 10(-5) s(-1) and 5.2 x 10(-6) s(-1) at 25 degrees C. The pattern of unfolding is not correlated with covalent heterogeneity, since a wide range of variants and site-directed mutants exhibit identical profiles, nor is the unfolding correlated with cis-trans Pro isomerization in the native state. In comparison with the apo form of cytochrome b(5), the kinetics of refolding and unfolding are more complex and exhibit very different transition states. The data support a model for unfolding in which heme-protein interactions give rise to two discernible rates of unfolding. From an analysis of the activation parameters associated with each process it is established that two structurally similar transition states differing by less than 5 kJ mol(-1) exist in the unfolding reaction. Protein refolding exhibits monophasic kinetics but with distinct curvature apparent in plots of ln k(obs) versus denaturant concentration. The data are interpreted in terms of alternative routes for protein folding in which a "fast track" leads to the rapid ordering of structure around Trp26 for refolding while a slower route requires additional reorganization around the hydrophobic core.

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 thermal stability of the tryptic fragment of bovine microsomal cytochrome b5 and a variant containing six additional residues

Thermally induced denaturation has been measured for both oxidised and reduced forms of the tryptic fragment or bovine microsomal cytochrome b 5 using spectrophotometric methods. In the oxidised state, the tryptic fragment of cytochrome b 5 (Ala 7-Lys 90) denatures in a single cooperative transition with a midpoint temperature ( T m) of ∼ 67°C (pH 7.0). The reduced form of the tryptic fragment of cytochrome b 5 shows a higher transition temperature of ∼ 73°C at pH 7.0 and this is reflected in the values of Δ H m, Δ S m, and Δ(Δ G) of ∼ 310kJ · mol −1, 900J · mol −1 · K −1 and 5 kJ · mol −1. Increased thermal stability is demonstrated for a variant protein that contains the first 90 amino acid residues of cytochrome b 5. These novel increases in stability are observed in both redox states and result from the presence of six additional residues at the amino-terminus. The two forms of cytochrome b 5 do not differ significantly in structure with the results suggesting that the reorganisation energy (λ) of the variant protein, as measured indirectly from redox-linked differences in conformational stability, is small. Consequently the reported subtle differences in reactivity between variants of cytochrome b 5 may result from the presence of additional N-terminal residues on the surface of the protein.

The role of the internal hydrogen bond network in first-order protein electron transfer between Saccharomyces cerevisiae iso-1-cytochrome c and bovine microsomal cytochrome b5.

An internal water molecule (designated WAT166) is found in iso-1-cytochrome c which is part of a redox-state-dependent hydrogen bond network. The position of this water molecule with respect to the polypeptide fold can be altered or even displaced by site-directed mutagenesis leading to structural perturbations and associated changes in redox potential. Using saturation transfer 1H-NMR methods, this study measures changes in the electron transfer reactivity for three variants of yeast iso-1-cytochromes c in which the position of this water molecule is altered. In particular, the reverse electron transfer rate is measured within a complex formed between either wild-type or variant yeast iso-1-cytochromes c and the tryptic fragment of bovine liver microsomal cytochrome b5. For three variants of yeast iso-1-cytochrome c the rate constants measured by saturation transfer are wild-type (Asn52, E0 = 270 mV, kex = 0.3 s-1), Asn52----Ala (E0 = 240 mV, kex = 0.6 s-1), Asn52----Ile (E0 = 220 mV, kex = 1.0 s-1). The first-order rates are compared with that of a fourth variant Phe82----Gly which has been measured previously (E0 = 220 mV, kex = 0.7 s-1). An analysis of the variation in the observed cross exchange rate using Marcus theory shows that these changes can be predicted quantitatively by the shift in redox potential that accompanies mutagenesis. So, although the perturbation of the internal water molecule by mutagenesis alters both the structure and redox potential of cytochrome c, surprisingly it does not significantly influence the intrinsic electron transfer reactivity of the protein. Studies of the activation parameters suggests that a variation of temperature changes both delta G* and also the prefactor. These data are discussed in terms of models involving dynamic molecular recognition between proteins.

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.