Hepatic Sulfite Oxidase

THE NATURE AND FUNCTION OF THE HEME PROSTHETIC GROUPS

From the ¹ From the Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27706

Sulfite oxidase (sulfite:O₂ oxidoreductase EC 1.8.3.1) was purified 1000-fold from acetone powdered bovine liver and shown to be 65 to 80% pure by several criteria including disc gel electrophoresis, sedimentation velocity, and gel exclusion chromatography.

The molecular weight of the enzyme was found to be 115,000 by the method of sedimentation equilibrium and it was shown to be composed of subunits, whose molecular weight was estimated by acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, and found to be 55,000. The rate of sedimentation of the enzyme in dilute phosphate buffer at 20° was 5.8 S.

The absorption spectrum of the enzyme exhibited a broad band centered at 530 nm with a shoulder at 560 nm and a sharp peak at 413 nm. Upon reduction of the enzyme with sulfite, absorption maxima were seen at 555, 526, 423, and 325 nm. The enzyme was found to contain heme which was quantitated as the pyridine-hemochromogen. Each molecule of enzyme contained 2 molecules of heme.

Several lines of evidence indicated that the heme was a prosthetic group of the enzyme. Heme content and sulfite oxidase activity were enriched in parallel during the purification procedure. Heme and sulfite oxidase activity migrated identically when subjected to disc gel electrophoresis and sedimentation in a centrifugal field. In addition, when the enzyme was heated, its heme lost the ability to be reduced by sulfite and there was a concurrent and proportional loss of sulfite oxidase activity.

The reduction of oxygen by sulfite oxidase exhibited an initially rapid phase which passed over into a slower and linear rate. This partial inactivation of the enzyme occurred by a first order process and correlated with reduction of half of the heme. When the sulfite was depleted, the heme reoxidized. When challenged with another aliquot of sulfite, the enzyme again exhibited the fast initial rate and the first order partial inactivation, thus demonstrating the complete reversibility of this phenomenon. The presence of sulfite did not change the sedimentation properties of sulfite oxidase.

The heme of sulfite oxidase was reduced in the steady state achieved in the simultaneous presence of sulfite and oxygen, but was oxidized in the steady state when the electron acceptor was ferricyanide.

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				R. Hansch, C. Lang, E. Riebeseel, R. Lindigkeit, A. Gessler, H. Rennenberg, and R. R. Mendel Plant Sulfite Oxidase as Novel Producer of H2O2: COMBINATION OF ENZYME CATALYSIS WITH A SUBSEQUENT NON-ENZYMATIC REACTION STEP J. Biol. Chem., March 10, 2006; 281(10): 6884 - 6888. [Abstract] [Full Text] [PDF]

				G. D'Errico, A. Di Salle, F. La Cara, M. Rossi, and R. Cannio Identification and Characterization of a Novel Bacterial Sulfite Oxidase with No Heme Binding Domain from Deinococcus radiodurans J. Bacteriol., January 15, 2006; 188(2): 694 - 701. [Abstract] [Full Text] [PDF]

				U. Kappler, B. Bennett, J. Rethmeier, G. Schwarz, R. Deutzmann, A. G. McEwan, and C. Dahl Sulfite:Cytochrome c Oxidoreductase from Thiobacillus novellus. PURIFICATION, CHARACTERIZATION, AND MOLECULAR BIOLOGY OF A HETERODIMERIC MEMBER OF THE SULFITE OXIDASE FAMILY J. Biol. Chem., April 28, 2000; 275(18): 13202 - 13212. [Abstract] [Full Text] [PDF]