Resonance Raman Characterization of Biotin Sulfoxide Reductase

Abstract

Resonance Raman spectroscopy has been used to define active site structures for oxidized Mo(VI) and reduced Mo(IV) forms of recombinant Rhodobacter sphaeroides biotin sulfoxide reductase expressed in Escherichia coli. On the basis of 18 With the exception of nitrogenase, molybdenum enzymes catalyze formal oxygen atom transfer between water and substrate and contain an active site in which the molybdenum is coordinated by the dithiolene side chain of one or two molybdopterins (Fig. 1a) (1-3). Although the recent proliferation of x-ray crystal structures for mononuclear molybdenum enzymes has revealed a common structure for the molybdopterin cofactor, they have also revealed considerable diversity in the molybdenum coordination environment (2-11). However, on the basis of the available crystallographic, spectroscopic, primary sequence, and cyanide inhibition data, these enzymes can be divided into three large families (Fig. 1b) (1). Hydroxylases, such as Desulfovibrio gigas aldehyde oxidoreductase, xanthine oxidase, and xanthine dehydrogenase, represent the xanthine oxidase family, characterized by a Mo(VI) active site with a single molybdopterin, as well as terminal oxo and sulfido groups. The oxotransferase class of molybdoenzymes catalyzes direct oxygen atom transfer between substrate and water and can be divided into two families. The sulfite oxidase family, as represented by sulfite oxidase and assimilatory nitrate reductase, contains Mo(VI) ligated by two oxo groups in addition to a single molybdopterin and a cysteine residue. The dimethyl sulfoxide (Me 2 SO) reductase family comprises mono-oxoMo(VI) sites ligated by two molybdopterins and a protein side chain ligand such as serine, cysteine, or selenocysteine. In addition to Me 2 SO reductase, this family also includes formate dehydrogenase, dissimilatory nitrate reductase, and biotin sulfoxide (BSO) 1 reductase. BSO reductase is found in bacterial systems, such as Rhodobacter sphaeroides and Escherichia coli, and catalyzes the reduction of d-biotin d-sulfoxide to d-biotin (Fig. 2). Although the precise role for BSO reductase in bacterial metabolism has yet to be defined, potential physiological functions include scavenging biotin sulfoxide from the environment, reducing bound intracellular biotin that has become oxidized in an aerobic environment, and protecting the cell from oxidative damage (12). Extensive characterization of this enzyme has been limited due to the low natural abundance of the protein and its constitutive expression (13). However, R. sphaeroides BSO reductase has recently been heterologously expressed in E. coli and characterized as containing the molybdopterin guanine dinucleotide form of the molybdopterin cofactor (14). Using either reduced methyl viologen or NADPH as electron donor, the enzyme was found to exhibit broad substrate specificity and was able to catalyze reduction of nicotinamide-N-oxide, methionine sulfoxide, trimethylamine-N-oxide, and dimethyl sulfoxide with varying efficiencies, in addition to the reduction of biotin ...

An Active Site Tyrosine Influences the Ability of the Dimethyl Sulfoxide Reductase Family of Molybdopterin Enzymes to Reduce S-Oxides

“…Reduction with Me 2 S-Whereas the ability of DMSOR to be reduced by Me 2 S has been studied extensively using various techniques (11,15,17), BSOR cannot be reduced by either Me 2 S or biotin (16), and the activity of TMAOR upon product addition has not been studied previously. To investigate how mutation of Tyr-114 alters this activity, data were obtained as described by Adams et.…”

“…Although there are some shifts in the cycled Mo(VI) absorption spectra of the mutants at longer wavelengths, all of the main features seen for the wild-type proteins still remain, indicating that the molybdenum coordination environments are similar. Resonance Raman studies have indicated that the absorption peak at 550 nm is associated with the Mo-O bond found in the hexa-coordinate, mono-oxo spectra (15,16). This absorption peak is changed very little from wild type in either of the Tyr mutants, and it does not appear that the presence or absence of this Tyr greatly perturbs the catalytic Mo-oxygen , and molybdenum concentration was determined using atomic absorption spectroscopy.…”

“…This process is reversible in DMSOR, and the ability of this enzyme to be reduced by dimethyl sulfide (Me 2 S) has been extensively studied (7,11,15,17). However, resonance Raman analysis has indicated that BSOR is unable to be reduced with either Me 2 S or by biotin (16). Other than mutation of the protein ligands to the molybdenum in DMSOR and BSOR (18,19), no mutagenic studies have been reported for TMAOR, BSOR, or Rhodobacter DMSOR, and there is little information about the roles of other amino acids in enzymatic activity.…”

“…2) (15,16). This process is reversible in DMSOR, and the ability of this enzyme to be reduced by dimethyl sulfide (Me 2 S) has been extensively studied (7,11,15,17).…”

“…Mechanistic studies are aided by the extensive structural information reported for this family, including several x-ray crystal structures (2, 4 -8), analysis by extended x-ray absorption fine structure spectroscopy (EXAFS) (9 -11), EPR (10 -14), and resonance Raman spectroscopy (15,16).…”