Zhong-Xian Huang

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

Structural and functional insights into polymorphic enzymes of cytochrome P450 2C8

The cytochrome P450 (CYP) superfamily plays a key role in the oxidative metabolism of a wide range of drugs and exogenous chemicals. CYP2C8 is the principal enzyme responsible for the metabolism of the anti-cancer drug paclitaxel in the human liver. Nearly all previous works about polymorphic variants of CYP2C8 were focused on unpurified proteins, either cells or human liver microsomes; therefore their structure–function relationships were unclear. In this study, two polymorphic enzymes of CYP2C8 (CYP2C8.4 (I264M) and CYP2C8 P404A) were expressed in E. coli and purified. Metabolic activities of paclitaxel by the two purified polymorphic enzymes were observed. The activity of CYP2C8.4 was 25% and CYP2C8 P404A was 30% of that of WT CYP2C8, respectively. Their structure–function relationships were systematically investigated for the first time. Paclitaxel binding ability of CYP2C8.4 increased about two times while CYP2C8 P404A decreased about two times than that of WT CYP2C8. The two polymorphic mutant sites of I264 and P404, located far from active site and substrate binding sites, significantly affect heme and/or substrate binding. This study indicated that two important nonsubstrate recognition site (SRS) residues of CYP2C8 are closely related to heme binding and/or substrate binding. This discovery could be valuable for explaining clinically individual differences in the metabolism of drugs and provides instructed information for individualized medication.

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 mechanism for heme to prevent Aβ1–40 aggregation and its cytotoxicity

The β-amyloid peptide (Aβ) aggregation in the brain, known as amyloid plaques, is a hallmark of Alzheimer’s disease (AD). The aberrant interaction of Cu2+ ion with Aβ potentiates AD by inducing Aβ aggregation and generating neurotoxic reactive oxygen species (ROS). In this study, the biosynthesized recombinant Aβ1–40 was, for the first time, used to investigate the mechanism for heme to prevent Aβ1–40 aggregation and its cytotoxicity. Cell viability studies of SH-SY5Y cells and rat primary hippocampal neurons showed that exogenous heme can protect the cells by reducing cytotoxicity in the presence of Cu2+ and/or Aβ1–40. UV–vis spectroscopy, circular dichroism spectroscopy, and differential pulse voltammetry were applied to examine the interaction between heme and Aβ1–40. It was proven that a heme–Aβ1–40 complex is formed and can stabilize the α-helix structure of Aβ1–40 to inhibit Aβ1–40 aggregation. The heme–Aβ1–40 complex possesses peroxidase activity and it may catalyze the decomposition of H2O2, reduce the generation of ROS downstream, and ultimately protect the cells. These results indicated that exogenous heme is able to alleviate the cytotoxicity induced by Aβ1–40 and Cu2+. This information may be a foundation to develop a potential strategy to treat AD.

Expression, Characterization, and Reaction of Recombinant Monkey Metallothionein-1 and Its C33M Mutant

After we modified the protocol of purification, monkey metallothionein-1 (mkMT-1) and its mutant at position 33 (C33M mutant) were efficiently expressed and purified by using the glutathione-S-transferase fusion protein system. The protein yield has been considerably improved (8 mg/L culture for mkMT-1 and 10 mg/L culture for C33M mutant). The recombinant MT-1 and C33M mutant were characterized by ESI-MS, UV, and CD spectra. The reactions of MI-1 and C33M mutant with 5,5′-dithiobis(2-nitrobenzoic acid) and EDTA also have been carefully studied. The pH titration of MT-1 and C33M mutant has been studied by UV and CD spectra. The mutation of cysteine-to-methionine at position 33 mostly maintains the α-domain structure similar to that in wild-type mkMT-1, but the C33M mutant has significant loss of stability and cooperative properties of the domain.

Converting Cytochrome b5 into Cytochrome c‐Like Protein

Cytochrome b5 (cyt b5) is a well-studied b-type cytochrome with heme bound noncovalently. Its heme-binding ability depends mostly on the strong axial ligation provided by residues His63 and His39 (Figure 1). Mutation studies on the axial ligands, aimed at creating new proteins with novel catalytic reactivity, have been limited by the fact that such alteration would lead to a substantial decrease in the heme-binding stability. Therefore, it would be of great interest to construct covalent linkages between heme and polypeptides so as to stabilize the heme prosthetic group, as found in c-type cytochromes, in which heme is covalently attached to a polypeptide chain via two thioether bonds between the heme vinyl groups and the cysteine residues of a classic Cys-Xaa-Xaa-CysHis (CXXCH) heme-binding peptide motif in the protein. By converting cyt b5 into a cyt c-like protein, it will be much easier to fulfill the functional conversion of cyt b5 mentioned above. Based on the wealth of molecular information obtained from X-ray crystallography studies of cyt b5 (PDB entry 1CYO), [5] we found out that residues Asn57 and Ser71 are close to the

The role of Thr5 in human neuron growth inhibitory factor

GIF, a member of the metallothionein (MT) family (assigned as MT3), is a neuron growth inhibitory factor that inhibits neuron outgrowth in Alzheimer’s disease. The conserved Thr5 is one of the main differences between GIF and other members in the MT family. However, natural sheep GIF has an unusual Ala5, casting doubt on the role of common Thr5. We constructed a series of human GIF mutants at site 5, and characterized their biochemical properties by UV spectroscopy, circular dichroism spectroscopy, EDTA reaction, 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) reaction, and pH titration. Their inhibitory activity toward neuron survival and neurite extension was also examined. Interestingly, the T5A mutant exhibited distinct metal thiolate activity in the EDTA and DTNB reactions, and also lost its bioactivity. Meanwhile, the T5S mutant had similar biochemical properties and biological activity as wild-type human GIF, indicating the hydroxyl group on the Thr5 was critical to the bioactivity of human GIF. We suggest the hydroxyl group in human GIF may help stabilize the biologically active conformation. On the other hand, lack of the hydroxyl group in sheep GIF may be partially compensated by its abnormal structure.

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.

Efficient expression of human soluble guanylate cyclase in Escherichia coli and its signaling-related interaction with nitric oxide

Soluble guanylate cyclase (sGC), as a nitric oxide (NO) sensor, is a critical heme-containing enzyme in NO-signaling pathway of eukaryotes. Human sGC is a heterodimeric hemoprotein, composed of a α-subunit (690 AA) and a heme-binding β-subunit (619 AA). Upon NO binding, sGC catalyzes the conversion of guanosine 5′-triphosphate (GTP) to 3′,5′-cyclic guanosine monophosphate (cGMP). cGMP is a second messenger and initiates the nitric oxide signaling, triggering vasodilatation, smooth muscle relaxation, platelet aggregation, and neuronal transmission etc. The breakthrough of the bottle neck problem for sGC-mediated NO singling was made in this study. The recombinant human sGC β1 subunit (HsGCβ619) and its truncated N-terminal fragments (HsGCβ195 and HsGCβ384) were efficiently expressed in Escherichia coli and purified successfully in quantities. The three proteins in different forms (ferric, ferrous, NO-bound, CO-bound) were characterized by UV–vis and EPR spectroscopy. The homology structure model of the human sGC heme domain was constructed, and the mechanism for NO binding to sGC was proposed. The EPR spectra showed a characteristic of five-coordinated heme-nitrosyl species with triplet hyperfine splitting of NO. The interaction between NO and sGC was investigated and the schematic mechanism was proposed. This study provides new insights into the structure and NO-binding of human sGC. Furthermore, the efficient expression system of E. coli will be beneficial to the further studies on structure and activation mechanism of human sGC.