Acid pH-induced conformational changes in bovine liver rhodanese.

The enzyme rhodanese is greatly stabilized in the range pH 4-6, and samples at pH 5 are fully active after several days at 23 degrees C. This is very different from results at pH greater than 7, where there is significant loss of activity within 1 h. A pH-dependent conformational change occurs below pH 4 in a transition centered around pH 3.25 that leads slowly to inactive rhodanese at pH 3 (t 1/2 = 22 min at pH3). The inactive rhodanese can be reactivated by incubation under conditions required for detergent-assisted refolding of denatured rhodanese. The inactive enzyme at pH 3 has the maximum of its intrinsic fluorescence spectrum shifted to 345 nm from 335 nm, which is characteristic of native rhodanese at pH greater than 4. At pH 3, rhodanese shows increased exposure of organized hydrophobic surfaces as measured by 1,1'-bis(4-anilino)naphthalene-5,5'-disulfonic acid binding. The secondary structure is maintained over the entire pH range studied (pH 2-7). Fluorescence anisotropy measurements of the intrinsic fluorescence provide evidence suggesting that the pH transition produces a state that does not display greatly increased average flexibility at tryptophan residues. Pepsin digestibility of rhodanese follows the pH dependence of conformational changes reported by activity and physical methods. Rhodanese is resistant to proteolysis above pH 4 but becomes increasingly susceptible as the pH is lowered. The form of the enzyme at pH 3 is cleaved at discrete sites to produce a few large fragments. It appears that pepsin initially cleaves close to one end of the protein and then clips at additional sites to produce species of a size expected for the individual domains into which rhodanese is folded. Overall, it appears that in the pH range between pH 3 and 4, titration of groups on rhodanese leads to opening of the structure to produce a conformation resembling, but more rigid than, the molten globule state that is observed as an intermediate during reversible unfolding of rhodanese.     

Acceptor substrate-potentiated inactivation of bovine liver rhodanese

The interaction of bovine liver rhodanese (thiosulfate:cyanide sulfurtransferase, EC 2.8.1.1) with the acceptor substrates, dithiothreitol or cyanide, was studied. When incubated in the presence of cyanide or dithiothreitol, rhodanese was inactivated in a time-dependent process. This inactivation was detectable only at low enzyme concentrations; the rate and degree of inactivation could be modulated by varying the substrate concentration or the system pH. Activity measurements and fluorescence spectroscopy techniques were used in examining the inactivation phenomenon. Sulfur transfer to dithiothreitol was measured by direct assay and was shown to involve the dequenching of enzymic intrinsic fluorescence that had been previously observed only with cyanide as the acceptor substrate. Substrate-potentiated inactivation of rhodanese (with cyanide) has been reported before, but the cause and nature of this interaction were unexplained. The results presented here are consistent with an explanation invoking oxidation of rhodanese in the course of inactivation.     

A study of the single polypeptide nature of rhodanese. A comparison of different preparations.

The enzyme rhodanese (EC 2.8.1.1) appears as a single polypeptide chain protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weight of this species is approx. 33 000. This contrasts with previous reports that rhodanese behaves on gel filtration chromatography as a rapidly equilibrating monomer-dimer system composed of identical subunits with a molecular weight of 18 500. We have investigated this apparent discrepancy by isolating the enzyme by the two different preparative procedures used in the above investigations. The two crystalline samples were subjected to gel filtration chromatography under a wide variety of conditions and to sodium dodecyl sulfate disc gel electrophoresis. The two preparations yielded rhodanese which behaved identically and no evidence for the monomeric species was obtained under any experimental condition tested. Thin-layer gel chromatography of clarified liver homogenates gave no evidence of rhodanese species other than that present in the purified samples. The variation in molecular weights observed in gel filtration chromatography may be a reflection of the conformational mobility of the enzyme leading to solvent-dependent changes in Stokes radius. If rhodanese is dimeric, special interactions must stabilize it under the conditions tested here.     

New crystalline derivatives of bovine liver rhodanese

Mitochondrial bovine liver rhodanese (thiosulfate:cyanide sulfurtransferase) has been crystallized in the form deprived of the transferable sulfur. The essential condition for crystallization was the removal of oxygen. Crystals of the sulfur-free enzyme are isomorphous with the previously characterized crystals of the sulfur-substituted enzyme. The new crystal species can react with either thiosulfate or selenosulfate to form the catalytic intermediate and, subsequently, with cyanide to form the corresponding product. Furthermore, the enzyme active site can be alkylated by iodoacetic acid.     

Immunological evidence for a conformational difference between recombinant bovine rhodanese and rhodanese purified from bovine liver

Rhodanese has been utilized as a model enzyme for the study of protein structure-function relationships. The enzyme has recently been cloned and the recombinant enzyme is now available for investigation. However, prior to use in structure-function studies, the recombinant enzyme must be shown to have the same structure and activity as the bovine liver enzyme used in the previous studies. An immunological study of the conformations of these enzyme conformers is described. Three antibodies (two monoclonal and one polyclonal, site-directed antibody) were shown to detect distinct and nonoverlapping epitopes. The epitopes of the monoclonal antirhodanese antibodies (R207 and MAB11) were mapped to the same CNBr digest fragment of the amino terminal domain of rhodanese, and the epitope of the site-directed antibody prepared against the interdomain tether sequence of rhodanese (PAT-T1) was mapped to that region of rhodanese (residues 142–156). The rhodanese conformers were studied by monitoring the accessibility of the epitopes recognized by each antibody in each conformer using an indirect ELISA. None of the antibodies could detect its epitope on the purified liver enzyme. Two of the antibodies (R207 and PAT-T1) could also not detect their epitopes on the recombinant enzyme. However, MAB11 did detect a conformational difference between the natural and recombinant rhodanese conformers, indicating the conformational difference is localized in the first 73 amino acids of rhodanese. This difference presumably reflects the difference in the histories of the two enzymes and may be due to differences in enzyme folding, differences in the purification procedures, and differences in storage conditions—all of which could influence the final conformation of the enzyme.     

Purification of bovine liver rhodanese by low-pH column chromatography

We report a purification of bovine liver rhodanese (thiosulfate:cyanide sulfurtransferase, EC 2.8.1.1) using column chromatography under conditions that take advantage of recent information regarding the structure and stability of this enzyme. At low pH (e.g., pH 4-6), rhodanese is stabilized against inactivation processes. By maintaining rhodanese at low pH, column chromatography, and especially ion-exchange chromatography, becomes practical, without loss of enzymatic activity. A purification method involving the sequential use of cation-exchange, size-exclusion, and hydrophobic-interaction chromatography was developed, and rhodanese was purified with good yield to electrophoretic purity and high specific activity. Previous methods for purifying bovine liver rhodanese employ repeated ammonium sulfate fractionations and crystallization of the rhodanese. In these methods, it is difficult to separate rhodanese from yellow-brown contaminants in the final stages of the procedures. Here, yellow-brown contaminants, which copurify with rhodanese on the first two columns, are completely resolved by hydrophobic interaction chromatography. This method can be readily scaled up, requires no special equipment, eliminates the variability inherent in previous methods, and is less dependent upon experience.     

The differential functional stability of various forms of bovine liver rhodanese

Bovine liver rhodanese (thiosulfate:cyanide sulfurtransferase, EC 2.8.1.1) was prepared in dilute solutions and subjected to conditions that led to a time-dependent loss of enzyme activity. The rate of this activity loss was found to be dependent upon the sulfur substitution state of the enzyme, and the presence or absence of the substrates, thiosulfate and cyanide. In the absence of excess substrates, free enzyme (E), and the covalent intermediate form of the enzyme bearing a divalent sulfur atom in the active site (ES), are of approximately equal functional stability. In comparison, E, in the presence of excess cyanide, was markedly more labile, while ES, supported by 10-50 mM thiosulfate, showed no significant loss of activity under any of the conditions tested. All the enzyme solutions were shown to be losing assayable protein from solution. However, it was demonstrated that, for rhodanese in the E form, the amount of protein lost was insufficient to account for the activity lost, and a marked decline in specific activity was observed. Enzyme in the ES form, whether supported by additional thiosulfate or not, did not decline in the specific activity, though comparable protein loss did occur from these solutions. Intrinsic fluorescence measurements of rhodanese in the ES form, before and after removal of the persulfide sulfur through the addition of cyanide, indicated that loss of enzymic activity was not accompanied by loss of the bound sulfur atom. Therefore, the stabilizing effect observed with thiosulfate could not be explained simply by its ability to maintain enzyme in the sulfur-substituted state. Since the concentration of thiosulfate employed in these experiments was insufficient to maintain all the enzyme in ES.S2O3 form, thiosulfate was acting as a chemical reagent rather than a substrate in stabilizing enzyme activity.     

Binding of metal cyanide complexes to bovine liver rhodanese in the crystalline state

Bovine liver rhodanese, which catalyzes the transfer of sulfur atoms between a variety of sulfur donor and sulfur acceptor substrates, is inhibited by metal cyanide complexes [Volini, M., Van Sweringen, B., & Chen, F.-Sh. (1978) Arch. Biochem. Biophys. 191, 205-215]. Crystallographic studies are described which reveal the binding mode of four different metal cyanides to bovine liver rhodanese: Na[Au(CN2], K2[Pt(CN)4], K2[Ni(CN)4], and K2[Zn(CN)4]. It appears that these complexes bind at one common site at the entrance of the active site pocket, interacting with the positively charged side chains of Arg-186 and Lys-249. This observation explains the inhibition of rhodanese by this class of compounds. For the platinum and nickel cyanide complexes virtually no other binding sites are observed. The gold complex binds, however, to three additional cysteine residues, thereby also displacing the extra sulfur atom which was bound to the essential Cys-247 in the sulfur-rhodanese complex. The zinc complex binds to completely different additional sites and forms complexes with the side chains of Asp-101 and His-203. Possible reasons for these different binding modes are discussed in terms of the preference for "hard" and "soft" ligands of these four metal ions.     

The structure of bovine liver rhodanese. II. The active site in the sulfur-substituted and the sulfur-free enzyme.

X-ray studies at 2.5 Å resolution show that the active site of bovine liver rhodanese is a depression between the two domains. In sulfur-substituted rhodanese the density of the essential Cys247 corresponds with that of a persulfide. Both sulfur atoms are interacting via hydrogen bonds with several peptide NH and side-chain OH groups. One side of the active site pocket contains mainly hydrophylic, the other side mainly hydrophobic residues. None of these hydrophylic or hydrophobic groups appears to interact strongly with the persulfide.Crystals of the sulfur-substituted enzyme were treated with cyanide, a sulfur acceptor. Subsequent difference Fourier studies show that the extra sulfur atom has been removed. Only minor conformational differences appear to exist between the two rhodanese species studied. These are a movement of the Sγ atom of Cys247 and some rearrangement of solvent molecules near the active site.The combination of these observations with the results of experiments performed by other investigators suggest a mechanism for sulfur transfer by rhodanese in which the thiol group of Cys247 is the essential nucleophile, whereas the positive charges on Arg186 and Lys249 act in various ways as “electrophilic assistants”. The transition state and the persulfide in the sulfur-substituted enzyme are stabilized by several hydrogen bonds.     

Detection of time-dependent and oxidatively induced antigens of bovine liver rhodanese with monoclonal antibodies

Rhodanese has been extensively utilized as a model protein for the study of enzyme structure-function relationships. An immunological study of conformational changes occurring in rhodanese as a result of oxidation or thermal inactivation was conducted. Three monoclonal antibodies (MABs) to rhodanese were produced. Each MAB recognized a unique epitope as demonstrated by binding of the MABs to different proteolytic fragments of rhodanese. While none of the MABs significantly bound native, soluble, sulfur-substituted bovine rhodanese, as indicated in indirect enzyme-linked immunosorbent assay experiments, each MAB was immunoadsorbed from solution by soluble rhodanese as a function of the time rhodanese was incubated at 37 degrees C. Thus, as rhodanese was thermally inactivated, conformational changes resulted in the expression of three new epitopes. Catalytic conformers demonstrated different rates of thermally induced antigen expression. Each MAB also recognized epitopes expressed when rhodanese was immobilized on microtiter plates at 37 degrees C. Two conformers resulting from oxidation of rhodanese by hydrogen peroxide were identified immunologically. All MABs recognized rhodanese that was oxidized with peroxide and allowed to undergo a secondary cyanide-dependent reaction which also resulted in a fluorescence shift and increased proteolytic susceptibility. Only one MAB was capable of recognizing an epitope expressed when rhodanese was oxidized with peroxide and then separated from the reactants to prevent induction of the secondary conformational changes.     

Expression of cloned bovine adrenal rhodanese

A cDNA for the enzyme rhodanese (thiosulfate:cyanide sulfurtransferase, EC 2.8.1.1) has been cloned from a bovine adrenal library. An initiator methionine codon precedes the amino-terminal amino acid found in the isolated protein. Rhodanese is synthesized in the cytoplasm and transferred to the mitochondrial matrix. Thus, any amino-terminal sequence required for organelle import is retained in the mature protein. Furthermore, the DNA sequence shows that there are three additional amino acids, Gly-Lys-Ala, at the carboxyl terminus that are not found by protein sequencing. Additionally, comparison of the published amino acid sequence with that encoded by the open reading frame revealed three differences in the amino acid sequence. Comparison of the bovine and chicken liver sequences shows an overall level of 70% sequence homology, but there is complete identity of all residues that have been implicated in the function of the enzyme. When two mammalian cells, cos-7 and 293 cells, were transiently transfected with a plasmid containing the rhodanese coding region, rhodanese activity in lysates increased approximately 20-fold. Fluorograms of denaturing polyacrylamide gels detected a large increase in a polypeptide that co-migrated with the native protein and reacted with anti-rhodanese antibodies. Nondenaturing gels showed two active species that co-migrated with the two major electrophoretic forms purified by current procedures. Escherichia coli, transformed with a plasmid containing the rhodanese coding region, showed a 15-fold increase in rhodanese activity over baseline values. When the E. coli recombinant protein was analyzed on a nondenaturing gel, only one species was observed that co-electrophoresed with the more electropositive variant seen in purified bovine liver rhodanese. This single variant could be converted by carboxypeptidase B digestion to a form of the enzyme that co-migrated with the more electronegative species isolated from bovine liver. Thus, two major, enzymatically active electrophoretic variants, commonly observed in mammalian cells, can be accounted for by carboxyl-terminal processing without recourse to other post-translational modifications.     

Cloning and sequence analysis of the human liver rhodanese: comparison with the bovine and chicken enzymes

The cDNA for the human rhodanese (thiosulfate: cyanide sulfurtransferase, EC 2.8.1.1), a nuclearly encoded protein of the mitochondrial matrix, was isolated from a human fetal liver cDNA library. Nucleotide sequence revealed an open reading frame coding for a polypeptide of 295 amino acids, which presented a 57% and 58% identity with the bovine and avian rhodanese, respectively. The analysis of the 5'-ends of the coding region gave no evidence for the presence of a cleavable signal sequence as found in other mitochondrial proteins. A comparison with two available amino acid sequences (cow and chicken) showed that sequence similarity is not restricted to the alpha-helices and beta-structures motifs which are remarkably superimposable in the two halves of bovine rhodanese, but extends to adjacent regions.