Yun-Hua Wang

The direct electrochemistry of cytochrome c in the presence of various amino acids

The promotion of horse heart cytochrome c electrochemistry at a gold minigrid electrode in the presence of some amino acids in solution has been investigated by cyclic voltammetry. Of these, Cys, His, Met, Cis and Trp have proved to be effective in facilitating electron transfer between Cyt c and the gold electrode, and good, quasi-reversible redox reactions were achieved. Under alkaline pH conditions, the above amino acids also present a promotion effect on the electrochemistry of Cyt c, but a different cyclic voltammogram appears. The new appearance results from the stability of different conformation states of Cyt c at different pH regions. Trp shows a more pH dependent ability in promoting the electrochemistry of Cyt c at a gold electrode. Probably, the proton dissociation and steric factor of the bulky indole group account for its electrochemical behavior. The spectroelectrochemistry of cytochrome c in the presence of various amino acids has also been examined

Crystal structure of recombinant trypsin-solubilized fragment of cytochrome b(5) and the structural comparison with Val61His mutant.

The crystal structure of the recombinant trypsin‐solubilized fragment of the microsomal cytochrome b5 from bovine liver has been determined at 1.9 Å resolution and compared with the reported crystal structure of the lipase‐solubilized fragment of the membrane protein cytochrome b5. The two structures are similar to each other. However, some detailed structural differences are observed: the conformation of the segment Asn16–Ser20 is quite different, some helices around the heme and some segments between the helices are shifted slightly, the heme is rotated about the normal of the mean plane of heme, one of the propionates of the heme exhibits a different conformation. The average coordination distances between the iron and the two nitrogen atoms of the imidazole ligands are the same in the two structures. Most of the structural differences can be attributed to the different intermolecular interactions which result from the crystal packing. The wild‐type protein structure is also compared with its Val61His mutant, showing that the heme binding and the main chain conformations are basically identical with each other except for the local area of the mutation site. However, when Val61 is mutated to histidine, the large side chain of His61 is forced to point away from the heme pocket toward the solvent region, disturbing the micro‐environment of the heme pocket and influencing the stability and the redox potential of the protein. Proteins 2000;40:249–257.

The effect of magnesium ion on the electrochemistry of cytochrome c and cytochrome b5 at a gold electrode modified with cysteine

Abstract The direct electrochemistry of cytochrome c, cytochrome b 5 and Cyt c/Cyt b 5 complex has been studied by cyclic voltammetry at a cysteine-modified gold electrode. The results indicate that this functional electrode permits selectively a quasi-reversible electrochemical response of cytochrome c only. The near-reversible electrochemical response of cytochrome b 5 was stimulated by Mg 2+ ion. From titration experiments, we observed that either the Mg 2+ ion or cytochrome b 5 could improve the cyclic voltammetric response of cytochrome c in different ways, and proved that the 1:1 protein–protein complex formed in the presence of Mg 2+ ion even at higher ionic strength. Meanwhile, we observed that the Mg 2+ ion and the ionic strength affected the electrochemical behaviour of cytochrome c more strongly than cytochrome b 5 . Thus, we propose that there are different interaction models involving protein–electrolyte–electrode between cytochrome c, cytochrome b 5 and the 1:1 protein complex with the electrode. The mutual interaction between two proteins was favourable for the heterogeneous electron transfer reaction, and the assumed interaction patterns of the 1:1 protein complex with the electrode were also confirmed by potential-step chronocoulometry.

The direct electrochemistry of cytochrome b5 and its mutant proteins

Abstract A near-reversible cyclic voltammetric response of cytochrome b 5 and its mutants, E44A, E56A and E44/56A, was observed at a gold electrode modified with thioglycolic acid. The electron transfer between the negatively charged protein and negatively charged electrode was promoted by the multivalent cations such as Mg 2+ or Cr(NH 3 ) 6 3+ ions. When the protein solution was titrated by Mg 2+ ion, the conditional reduction potential E O′ shifted to the positive direction and reached a plateau after Mg 2+ ion was more than 20 mM. Under this condition, the E O′ of wild type cytochrome b 5 and its mutants are −6 mV (vs. SHE, wild type Cyt b 5 ), −7 mV (Cyt b 5 E44A), −2 mV (Cyt b 5 E56A), −3 mV (Cyt b 5 E44/56A) respectively. The conditional reduction potentials of cytochrome b 5 and its mutants obtained by spectroelectrochemical titration in the presence of 0.01 mM Ru(NH 3 ) 6 3+ , pH 7.0, I = 0.1 M phosphate buffer are +5 mV (vs. SHE, wild type Cyt b 5 ), +6 mV (Cyt b 5 344A), +6 mV (Cyt b 5 E56A), + 7 mV (Cyt b 5 E44/56A) respectively. The results demonstrate that the mutagenesis at surface residues Glu44 and Glu56 does not alter the reduction potential of cytochrome b 5 significantly. However, the studies on binding between cytochrome b 5 and Mg 2+ ion by the electrochemistry and NMR show that although the Mg 2+ ion has the same interaction with these negatively charged residues and similar structural perturbation, the chelation of Mg 2+ ions by the heme propionate appears a stronger influence on the heme, shifting the reduction potential of proteins.

Redox properties of cytochrome b5: a mutagenesis and DPV study of the pH and ionic strength dependence of redox potentials and interactions with myoglobin by DPV

To understand the determinants of redox potentials in cytochrome b5 (cyt b5), wild type cyt b5 and its E44A, E56A, E44/56A and F35Y mutants have been characterized. The pH and ionic strength dependence of the redox potentials of cyt b5 and its mutants have been investigated by differential pulse voltammetry (DPV). Variation of pH demonstrated that there are ionizable functional groups that are linked to the redox potential of cyt b5. The Glu44 and Glu56 mutants produced the highest pK values. The F35Y mutant had the lowest pK . These results imply that there are different rearrangements of the charge density distributed in the hydrogen bond networks (involving Glu44 and Glu56) as well as in the aromatic network (involving Phe35). Analysis of the diffusion coefficients Do and the heterogeneous electron transfer rate constants k o ?, demonstrate that the acidic residues Glu44 and Glu56 participate in interaction not only with their partner proteins, but also with the surface of the electrode. The residue Phe35 plays an important role in regulating the redox potentials of cyt b5. The results of both the direct electrochemistry and the protein � /protein pH titration imply that the cyt b5jmyoglobin interface has both electrostatic and hydrophobic interactions. # 2002 Elsevier Science B.V. All rights reserved.

Effects of charged amino‐acid mutation on the solution structure of cytochrome b5 and binding between cytochrome b5 and cytochrome c

The solution structure of oxidized bovine microsomal cytochrome b5 mutant (E48, E56/A, D60/A) has been determined through 1524 meaningful nuclear Overhauser effect constraints together with 190 pseudocontact shift constraints. The final family of 35 conformers has rmsd values with respect to the mean structure of 0.045±0.009 nm and 0.088±0.011 nm for backbone and heavy atoms, respectively. A characteristic of this mutant is that of having no significant changes in the whole folding and secondary structure compared with the X‐ray and solution structures of wild‐type cytochrome b5. The binding of different surface mutants of cytochrome b5 with cytochrome c shows that electrostatic interactions play an important role in maintaining the stability and specificity of the protein complex formed. The differences in association constants demonstrate the electrostatic contributions of cytochrome b5 surface negatively charged residues, which were suggested to be involved in complex formation in the Northrup and Salemme models, have cumulative effect on the stability of cyt c‐cyt b5 complex, and the contribution of Glu48 is a little higher than that of Glu44. Moreover, our result suggests that the docking geometry proposed by Northrup, which is involved in the participation of Glu48, Glu56, Asp60, and heme propionate of cytochrome b5, do occur in the association between cytochrome b5 and cytochrome c.

The effect of mutation at valine-45 on the stability and redox potentials of trypsin-cleaved cytochrome b5

In an attempt to elucidate the determinants of redox potential and protein stability in cytochrome b5, three mutants at a highly conserved residue Val45, which is a member of heme hydrophobic pocket residues have been characterized. The V45Y mutant was designed to introduce a bulkier residue and a hydroxyl group to the heme pocket. The mutants V45H and V45E were constructed to test the effect of positive and negative charge on the stability and redox potential of proteins. The influence of these mutants on the protein stability towards thermal, urea, acid, ethanol and on the redox potential were studied. It is concluded that the decrease of hydrophobic free energy and the larger volume of the tyrosine make the phenylhydroxyl group of tyrosine still sitting inside the hydrophobic pocket, while the side chain of the mutant V45E and V45H shift away from the heme pocket. The redox potentials of mutants V45Y, V45H, V45E and wild-type of cytochrome b5 are -35 mV, 8 mV, -26 mV and -3 mV, respectively. The bigger change of the V45Y on redox potential is due to the close contact between the hydroxyl group and the heme, while the changes of the V45E and V45H result from the alteration of charge density and distribution around the heme. Different relative stability of these mutants towards heat have been observed with the order: WT > V45Y-V45H > V45E being both in the oxidized and reduced state. The relative stability induced by addition of urea decreases in the order: WT > V45Y > V45H > V45E. These results suggest that the difference in the hydrophobic free energy is a major factor contributing to the stability of the Val45 mutants. Also the loose of the helix III in the mutant V45E makes it more unstable. These results indicate that residue Val45 plays an important role in the stability and redox potential of the protein.

The comparative study on the solution structures of the oxidized bovine microsomal cytochrome b5 and mutant V45H.

A comparative study on the solution structures of bovine microsomal cytochrome b5 (Tb5) and the mutant V45H has been achieved by 1D and 2D 1H‐NMR spectroscopy to clarify the differences in the solution conformations between these two proteins. The results reveal that the global folding of the V45H mutant in solution is unchanged, but the subtle changes exist in the orientation of the axial ligand His39, and heme vinyl groups. The side chain of His45 in V45H mutant extends to the outer edge of the heme pocket leaving a cavity at the site originally occupied by the inner methyl group of Val45 residue. In addition, the imidazole ring of axial ligand His39 rotates counterclockwise by ∼3° around the His‐Fe‐His axis, and the 4‐heme vinyl group turns to the space vacated by the removed side chain due to the mutation. Furthermore, the helix III of the heme pocket undergoes outward displacement, while the linkage between helix II and III is shifted leftward. These observations are not only consistent with the pattern of the pseudocontact shifts of the heme protons, but also well account for the lower stability of V45H mutant against heat and urea.

Spectroelectrochemical studies of cytochrome b5 Phe35 mutants

Reduction potentials of wild type and Phe35Tyr, Phe35His, Phe35Leu mutants of cytochrome b5 were studied using a spectroelectrochemical method, and a 2D 1H NMR study of a Phe35Tyr mutant was also performed to investigate the structural influence of Phe35 on the redox potential of cytochrome b5. All the Phe35Tyr, Phe35Leu and Phe35His mutant proteins exhibit a decreased redox potential in the order Phe35Tyr<Phe35His<Phe35Leu<wild type. The difference in E°′ is about 70 mV between Phe35Tyr and the wild type protein. Spectroelectrochemistry and structural studies demonstrated that the mutation at the Phe35 site perturbed the hydrophobicity of the heme pocket of cytochrome b5 seriously, leading to a considerable shift of the redox potential. The wild type and mutant cytochrome b5 showed increased redox potentials with increase of ionic strength, but the redox potential did not alter significantly with different mediators such as [Ru(NH3)6]Cl3, K3[Fe(CN)6], [Ru(NH3)5BzIm]Cl3 or cytochrome c present in the solution.

METHANOL-INDUCED UNFOLDING AND REFOLDING OF CYTOCHROME B5 AND ITS P40V MUTANT MONITORED BY UV-VISIBLE, CD, AND FLUORESCENCE SPECTRA

In order to illustrate the structural importance of proline-40 of cytochrome b5 (Cyt b5), the P40V mutant gene was constructed. Unfolding and refolding of Cyt b5 induced by methanol was investigated by means of the UV-visible spectrum, circular dichroism, and the fluorescence spectrum. Methanol denaturation of Cyt b5 is a cooperative process, that is, the heme group dissociates from the heme pocket accompanied by unfolding of the polypeptide chain both in the secondary and tertiary structures. Substitution of proline by valine reduces the stability of the mutant under methanol denaturation. The unfolding process is almost reversible by dilution. During refolding, the denatured polypeptide must be folded to a more ordered structure prior to the heme capture. Pro40 plays an important role in modulating the proteins stability. The role of tyrosine in the unfolding and refolding of Cyt b5 is evaluated for the first time. A mechanism of methanol denaturation is also proposed.