Site-directed Mutagenesis of Recombinant Sulfite Oxidase

Abstract

Each of the four cysteines in rat sulfite oxidase was altered by site-directed mutagenesis to serine, and the mutant proteins were expressed in Escherichia coli. Three of the replacements proved to be silent mutations, while a single cysteine, Cys-207, was found to be essential for enzyme activity. The C207S mutation was also generated in cloned human sulfite oxidase. The mutant human enzyme also displayed severely attenuated activity but was expressed at higher levels allowing purification and spectroscopic analysis. The absorption spectrum of the isolated molybdenum domain of the human C207S mutant displayed marked attenuation of the peak at 350 nm and a lesser decrease in absorbance from 450-600 nm as compared with the native human molyb-denum domain. The molybdenum and molybdopterin contents of the two samples were comparable. These data suggest that the major features in the absorption spectrum of the native molybdenum domain arise from the binding of Cys-207 to the molybdenum and indicate that this residue functions as a ligand of the metal. Sulfite oxidase, located in the intermembrane space of animal mitochondria, catalyzes the oxidation of sulfite to sulfate, the terminal reaction in the oxidative degradation of the sulfur-containing amino acids, cysteine and methionine. The enzyme is a dimer of identical subunits of mass 52 kDa. The N-terminal domain of mass 10 kDa forms a b 5-type cytochrome, and the C-terminal domain of mass 42 kDa anchors the molybdenum cofactor. The molybdenum cofactor in sulfite oxidase consists of molybdopterin (MPT), 1 a 6-alkyl-dihydropterin containing a unique cis-dithiolene moiety coordinated to molybdenum (1). EXAFS studies of rat liver sulfite oxidase have provided evidence for the presence of 2 MoO, 2 to 3 Mo-S, and 1 Mo-O(N) bonds at the molybdenum center (2, 3). The complete amino acid sequences of sulfite oxidase from chicken (4), rat (5), and human (6) sources have been reported. In addition, the amino acid sequences of a related enzyme nitrate reductase have been reported from a variety of fungal and plant sources (9-17). 2, 3 Nitrate reductase catalyzes the reduction of nitrate to nitrite, a critical reaction in the nitrogen assimilation pathway in fungi and higher plants. The enzyme contains three prosthetic groups: the molybdenum cofactor, a b 557 cytochrome, and FAD in binding domains encoded by distinct segments of the primary sequence. Unlike in sulfite oxidase, the molybdenum domain of nitrate reductase is at the N terminus, followed by the central heme domain and the C-terminal flavin domain. The amino acid sequences of the molybdenum domains of sulfite oxidase and nitrate reductase are approximately 37% identical, and a single cysteine residue, corresponding to Cys-207 of sulfite oxidase, is invariant in all of the sulfite oxidases and nitrate reductases sequenced to date. It has been postulated that this cysteine functions as a ligand to molybdenum (4, 6). Recently it was shown that mutation of the corresponding cysteine residue in nitrate reductase leads to loss of activity (18). This report describes the site-directed mutagenesis of rat and human sulfite oxidase to generate cysteine to serine mutants for each of the four cysteines in sulfite oxidase. The molybdenum domain of the human sulfite oxidase C207S mutant has been purified, and spectroscopic data indicate that Cys-207 functions as a ligand of molybdenum. MATERIALS AND METHODS Chemicals and Reagents-All common reagents were from Sigma and of the highest grade available. Premixed cell culture media were obtained from Life Technologies, Inc. Restriction enzymes were purchased from Stratagene, Life Technologies, Inc., or Amersham Corp. Escherichia coli host strain JM109 was purchased from Stratagene. Expression vector pPROK1, E. coli host strain DH5-, and reagents for site-directed mutagenesis were purchased from Clontech. Reagents for DNA sequencing were obtained from Amersham. Molybdenum Quantitation-Molybdenum was measured using a Perkin-Elmer Zeeman/3030 atomic absorption spectrophotometer equipped with autosampler model AS-60. All buffers and standard solutions were made using double-deionized water. Samples were equil-ibrated in 50 mM Tris acetate, pH 8.0, mixed with an equal volume of concentrated HNO 3 , wet ashed at 100 °C for 6 h, and diluted in 0.2% HNO 3 to an approximate molybdenum concentration of 10 ppb. A standard curve was obtained using solutions of 0, 5, and 15 ppb molybdenum in 0.2% HNO 3. All other conditions were as described by Johnson (19). Strains and Growth Conditions of E. coli-All plasmids were maintained in E. coli strain JM109 or DH5-. Cultures were grown in LB medium (Life Technologies, Inc.) containing 100 g/ml ampicillin at 37 °C. Cultures of E. coli strain DH5-for the expression of recombi-nant sulfite oxidase were grown in 2 liters of LB medium containing 100 g/ml carbenicillin for 16 h at 37 °C, subcultured into 30 liters of LB medium containing 100 g/ml ampicillin, 50 M sodium molybdate, 5 ml of Antifoam A, 0.4 mM isopropyl-D-thiogalactopyranoside and grown for 6 h at 37 °C. Purification of Sulfite Oxidase-All buffers used in the purification procedure contained 0.5 mM EDTA and 0.1 mg/ml phenylmethylsulfo-nyl fluoride. Cells from a 30-liter culture were harvested by centrifu-gation and resuspended in 10 mM potassium phosphate, pH 7.8. The cell pellet was weighed and resuspended in 1 volume of 50 mM potassium phosphate, pH 7.8 (phosphate buffer), and the cells were disrupted by