Effects of large-scale amino acid substitution in the polypeptide tether connecting the heme and molybdenum domains on catalysis in human sulfite oxidase

Sulfite oxidase (SO) is a molybdenum-cofactor-dependent enzyme that catalyzes the oxidation of sulfite to sulfate, the final step in the catabolism of the sulfur-containing amino acids, cysteine and methionine. The catalytic mechanism of vertebrate SO involves intramolecular electron transfer (IET) from molybdenum to the integral b-type heme of SO and then to exogenous cytochrome c. However, the crystal structure of chicken sulfite oxidase (CSO) has shown that there is a 32 ? distance between the Fe and Mo atoms of the respective heme and molybdenum domains, which are connected by a flexible polypeptide tether. This distance is too long to be consistent with the measured IET rates. Previous studies have shown that IET is viscosity dependent (Feng et al., Biochemistry, 2002, 41, 5816) and also dependent upon the flexibility and length of the tether (Johnson-Winters et al., Biochemistry, 2010, 49, 1290). Since IET in CSO is more rapid than in human sulfite oxidase (HSO) (Feng et al., Biochemistry, 2003, 42, 12235) the tether sequence of HSO has been mutated into that of CSO, and the resultant chimeric HSO enzyme investigated by laser flash photolysis and steady-state kinetics in order to study the specificity of the tether sequence of SO on the kinetic properties. Surprisingly, the IET kinetics of the chimeric HSO protein with the CSO tether sequence are slower than wildtype HSO. This observation raises the possibility that the composition of the non-conserved tether sequence of animal SOs may be optimized for individual species.     

Amino acid sequence of the 'b5-like' heme-binding domain from chicken sulfite oxidase

We present in this paper the sequence of the heme-binding domain of chicken sulfite oxidase which can be obtained by chymotryptic digestion of the native enzyme. The results of an automatic degradation have been reported previously. In the present work peptides were obtained from the heme-binding domain by digestion with trypsin, chymotrypsin and Staphylococcus aureus V8 protease; they were manually sequenced by the dansyl/Edman procedure. The evidence thus obtained is sufficient to completely establish the order of the 97 residues. In addition, two rounds of Edman degradation on sulfite oxidase itself allowed us to identify the same two residues, H-Ala-Pro, present at the N-terminus of the heme-binding domain; this result suggests that the latter constitutes the amino-terminal end of the sulfite oxidase peptide chain. The data presented here confirm the strong similarity between sulfite oxidase and microsomal cytochrome b5 already suggested by our first results. A sequence alignment is proposed for the two proteins. Inspection of the calf liver cytochrome b5 three-dimensional model together with the alignment suggests a similar overall structure for sulfite oxidase core with a limited number of backbone modifications. Our results point to a common evolutionary origin for sulfite oxidase core and microsomal cytochrome b5.     

The kinetic behavior of chicken liver sulfite oxidase.

A comprehensive kinetic study of sulfite oxidase has been undertaken over the pH range 6.0-10.0, including conventional steady-state work as well as rapid kinetic studies of both the reaction of oxidized enzyme with sulfite and reduced enzyme with cytochrome c (III). A comparison of the pH dependence of kcat, kred, and kox indicates that kred is principally rate limiting above pH 7, but that below this pH the pH dependence of kcat is influenced by that of kox. The pH independence of kred is consistent with our previous proposal concerning the reaction mechanism, in which attack of the substrate lone pair of electrons on a Mo(VI)O2 unit initiates the catalytic sequence. The pH dependence of kred/Kdsulfite indicates that a group on the enzyme having a pKa of approximately 9.3 must be deprotonated for effective reaction of oxidized enzyme with sulfite, possibly Tyr 322, which from the crystal structure of the enzyme constitutes part of the substrate binding site. There is no evidence for the HSO3-/SO32- pKa of approximately 7 in the pH profile for kred/Kdsulfite, suggesting that enzyme is able to oxidize the two equally well. By contrast, kcat/Kmsulfite and kred/Kdsulfite exhibit distinct pH dependence (the former is bell-shaped, the latter sigmoidal), again consistent with the oxidative half-reaction contributing to the kinetic barrier to catalysis at low pH. The pH dependence of kcat/Km(cyt c) (reflecting the second-order rate of reaction of free enzyme with free cytochrome) is bell-shaped and closely resembles that of kox/Kd(cyt c), reflecting the importance of the oxidative half-reaction in the low substrate concentration regime. The pH profile for kox/Kd(cyt c) indicates that two groups with a pKa of approximately 8 are involved in the reaction of free reduced enzyme with cytochrome c, one of which must be deprotonated and the other protonated. These results are consistent with the known electrostatic nature of the interaction of cytochrome c with its physiological partners.     

Conserved amino acid sequence domains in alpha-amylases from plants, mammals, and bacteria

Although alpha-amylases from mammals, plants, and bacteria have common functions, the amino acid sequences of enzymes from these three, evolutionarily distant groups of organisms are not known to share common homologies, and active sites have not been identified. Here I demonstrate that there are three sequence domains common to all alpha-amylases that are aligned and spaced at similar intervals along the length of each protein. The first domain in the barley enzymes appears to contain a calcium binding site. These common domains may represent important functional regions, perhaps the active sites.     

Primary structure of chicken pituitary prolactin deduced from the cDNA sequence. Conserved and specific amino acid residues in the domains of the prolactins

The perform of chicken prolactin (PRL) deduced from the cDNA sequence contains a signal peptide of 30 amino acid residues followed by a mature PRL of 199 residues. Chicken PRL shows 77, 68, 67, 58, and 31% identity of amino acid sequence with whale, human, ovine, rat, and salmon PRLs, respectively. Elucidation of the primary structure of avian PRL enabled extended analysis of the specific and conserved amino acid residues and domains of the PRL molecules. The mammalian, teleostean, and avian PRLs share 32 common residues, and these conserved residues are observed to cluster in four distinct domains (PD1 to PD4), corresponding to four of five conserved domains of the growth hormones. Of the 32 residues, 8 residues in the PD2 and PD4 domains, including 4 cysteines, are conserved by other members of the growth hormone family, which indicates that these 8 residues may be essential for common structural features of the gene family. On the other hand, 13 other residues distributed among all four domains are conserved almost exclusively in the PRLs, suggesting that these residues are indispensable for specific binding of the PRLs to their receptors.     

The complete amino acid sequence of chicken skeletal-muscle enolase.

The complete amino acid sequence of chicken skeletal-muscle enolase, comprising 433 residues, was determined. The sequence was deduced by automated sequencing of hydroxylamine-cleavage, CNBr-cleavage, o-iodosobenzoic acid-cleavage, clostripain-digest and staphylococcal-proteinase-digest fragments. The presence of several acid-labile peptide bonds and the tenacious aggregation of most CNBr-cleavage fragments meant that a commonly used sequencing strategy involving initial CNBr cleavage was unproductive. Cleavage at the single Asn-Gly peptide bond with hydroxylamine proved to be particularly useful. Comparison of the sequence of chicken enolase with the two yeast enolase isoenzyme sequences shows that the enzyme is strongly conserved, with 60% of the residues identical. The histidine and arginine residues implicated as being important for the activity of yeast enolase are conserved in the chicken enzyme. Secondary-structure predictions are analysed in an accompanying paper [Sawyer, Fothergill-Gilmore & Russell (1986) Biochem. J. 236, 127-130].     

Subcellular localisation of guinea pig hepatic molybdenum hydroxylases.

Molybdenum hydroxylase activity in guinea pig liver has been compared with that of marker enzymes in mitochondria (succinate dehydrogenase), microsomes (glucose-6-phosphatase) and cytosol (lactate dehydrogenase). Aldehyde oxidase activity was highest in the cytosol, with about 10-fold activity of xanthine oxidase. Significant molybdenum hydroxylase activity was found in mitochondria with minimal levels in microsomes. Mitochondrial and cytosolic aldehyde oxidase varied in substrate specificity and electrophoretic mobility with two major bands in each fraction, one of which was common to cytosol and mitochondria.     

Tryptic cleavage of rat liver sulfite oxidase. Isolation and characterization of molybdenum and heme domains.

Treatment of rat liver sulfite oxidase with trypsin leads to loss of ability to oxidize sulfite in the presence of cytochrome c as electron acceptor. Ability to oxidize sulfite with ferricyanide as acceptor is undiminished, while sulfite leads to O2 activity is partially retained. Gel filtration of the proteolytic products has led to the isolation of two major fragments of dissimilar size derived from sulfite oxidase. The smaller fragment has a molecular weight of 9500 and appears to be monomeric when detached from sulfite oxidase. It contains the heme in its cytochrome b5 structure, has no sulfite oxidase activity, and is reducible with dithionite but not with sulfite. The heme fragment can mediate electron transfer between pig liver microsomal NADH cytochrome b5 reductase and cytochrome c. The larger fragment has a molecular weight of 47,400 under denaturing conditions but elutes from Sephadex G-200 as a dimer. It contains no heme but retains all of the molybdenum and the modified sulfite-oxidizing capacity present in the proteolytic mixture. All of the EPR properties of the molybdenum center of native sulfite oxidase are retained in the molybdenum fragment. The molybdenum center is a weak chromophore with an absorption sectrum suggestive of coordination with sulfur ligands. Reduction by sulfite generates a spectrum attributable to molybdenum (V). Spectra of oxidized and sulfite-reduced preparations are sensitive to anions and pH. NH2-terminal analysis of native sulfite oxidase and the two tryptic fragments has permitted the conclusion that the sequence represented by the heme fragment is the NH2 terminus of native enzyme. These studies have demonstrated that the two cofactor moieties of sulfite oxidase are contained in distinct domains which are covalently held in contiguity by means of an exposed hinge region. Isolation of functional heme and molybdenum domains of sulfite oxidase after tryptic cleavage has demonstrated conclusively that the cytochrome b5 region of the molecule is required for electron transfer to the physiological acceptor, cytochrome c.     

Radiation inactivation of hepatic sulfite oxidase

Sulfite oxidase (EC 1.8.3.1), purified from chicken liver, is comprised of two identical subunits of 55 kDa, each of which contains a molybdenum and heme prosthetic group. The functional size of sulfite oxidase was determined by radiation inactivation analysis using both full, sulfite:cytochrome c reductase, and partial, sulfite:ferricyanide reductase, catalytic activities. Inactivation of full enzyme activity indicated a target size of 42 kDa while the partial activity indicated a target size of 25 kDa. These results confirm the earlier findings of two equivalent subunits and suggest the presence of a functional domain within the subunit structure that contains the molybdenum center and exhibits a smaller molecular mass than that of the enzyme subunit.     

Role of mammalian cytosolic molybdenum Fe-S flavin hydroxylases in hepatic injury

The study was designed to investigate the role of molybdenum iron-sulfur flavin hydroxylases in the pathogenesis of liver injuries induced by structurally and mechanistically diverse hepatotoxicants. While carbon tetrachloride (CCl4), thioacetamide (TAA) and chloroform (CHCl3) inflict liver damage by producing free radicals, acetaminophen (AAP) and bromobenzene (BB) exert their effects by severe glutathione depletion. Appropriate doses of these compounds were administered to induce liver injury in rats. The activities of the Mo-Fe-S flavin hydroxylases were measured and correlated with the biochemical markers of hepatic injury. The activity levels of the anti-oxidative enzymes and glutathione redox cycling enzymes were also determined. The treatment of rats with the hepatotoxins that inflict liver injury by generating free radicals (CCl4, TAA, CHCl3) had elevated activity levels of hepatic Mo-Fe-S flavin hydroxylases (p<0.05). Specific inhibition of these hydroxylases by their common inhibitor, sodium tungstate, suppresses biochemical and oxidative stress markers of hepatic tissue damage. On the contrary, Mo-Fe-S flavin hydroxylases did not show any change in animals receiving AAP and BB. Correspondingly, sodium tungstate could not attenuate damage in AAP and BB treated groups of rats. The study concludes that Mo-Fe-S hydroxylases contribute to the hepatic injury inflicted by free radical generating agents and does not play any role in hepatic injury produced by glutathione depleting agents. The study has implication in understanding human liver diseases caused by a variety of agents, and to investigate the efficacy of the inhibitors of Mo-Fe-S flavin hydroxylases as potential therapeutic agents.     

Deduced amino acid sequence of mature chicken testis ferredoxin

The cDNA sequence encoding the complete mature form of the steroidogenic ferredoxin from chicken testis has been determined and the amino acid sequence deduced therefrom has been compared with the sequences of bovine, human and porcine steroidogenic ferredoxins. The chicken sequence is between 84% and 88% identical with those of the other mitochondrial iron-sulfur proteins. Thus, the amino acid structure of steroidogenic ferredoxins which transfer electrons to mitochondrial forms of cytochrome P-450 has been very highly conserved over evolutionary time.     

The covalent structure of hepatic microsomal epoxide hydrolase. II. The complete amino acid sequence

The complete amino acid sequence of hepatic microsomal epoxide hydrolase has been determined. The protein contains 455 amino acid residues in a single polypeptide chain and has Mr = 52,691. Peptides from selective chemical and proteolytic cleavages were isolated by a combination of gel filtration and high performance liquid chromatography and sequenced by automated Edman degradation. Overlapping peptide sequences were used to deduce the complete sequence. This is the first epoxide hydrolase and the third microsomal enzyme for which the complete sequence has been determined.     

Studies on cytochrome c oxidase, VIII. The amino acid sequence of polypeptide VII.

The sequence determination of polypeptide VII from beef heart cytochrome c oxidase is described. The amino acid sequence is deduced from overlapping tryptic peptides and peptides obtained after cleavage with Staphylococcus aureus protease. The protein consists of 85 amino acids corresponding to a Mr of 10026, in agreement with a value of 9500 obtained by sodium dodecyl sulfate gel electrophoresis. The amino acid sequence around the only methionine present is very similar to sequences around the invariant heme binding methionine of the cytochrome c family. This similarity suggests that the protein is one of the heme bindings subunits of the oxidase.     

The structure of the molybdenum cofactor. Characterization of di-(carboxamidomethyl)molybdopterin from sulfite oxidase and xanthine oxidase

A di-(carboxamidomethyl) derivative of molybdopterin, the organic component of the molybdenum cofactor, has been prepared under conditions favoring retention of all of the structural features of the molecule. The specific radioactivity of [1-14C]iodoacetamide incorporated relative to the amount of phosphate indicated two alkylation sites per pterin. Energy-dispersive x-ray analysis of the derivative showed the presence of 2 sulfurs in the derivative. An exact mass corresponding to the molecular formula C14H18N7O5S2 was obtained for the MH+ ion of the alkylated, dephosphorylated compound by fast atom bombardment mass spectroscopy. 1H NMR spectra of the phosphorylated and dephosphorylated forms of alkylated molybdopterin, in conjunction with the other data, have provided strong corroboration of the validity of the proposed structure of molybdopterin (Johnson, J. L., and Rajagopalan, K. V. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 6856-6860) as a 6-alkylpterin with a 4-carbon side chain containing an enedithiol on C-1' and C-2', a secondary alcohol on C-3', and a phosphorylated primary alcohol on C-4'. As isolated, the di-(carboxamido-methyl)molybdopterin was found to be a 5,6,7,8-tetrahydropterin.     

Purification and amino acid sequence of chicken liver cathepsin L

Cathepsin L was purified from chicken liver lysosomes by a two-step procedure. Cathepsin L exhibited a single band of Mr 27,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions, presented a high affinity for the substrate Z-Phe-Arg-NMec, was very unstable at neutral pH, and was inhibited by Z-Phe-Phe-CHN2. The complete amino acid sequence of cathepsin L has been determined and consists of 215 residues. The sequence was deduced from analysis of peptides generated by enzymatic digestions and by chemical cleavage at methionyl bonds. Comparison of the amino acid sequence of cathepsin L with those of rat liver cathepsins B and H and papain demonstrates a striking homology among their primary structures.