Structure of the Molybdenum Site of Dimethyl Sulfoxide Reductase

George, Graham N.; Hilton, J.; Temple, Carrie A.; Prince, Roger C.; Rajagopalan, K.V.

doi:10.1021/ja982843k

Cited by 160 publications

(265 citation statements)

References 60 publications

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“…Since a single turnover converts the isolated, recombinant enzyme to the native state and form, the difference between the two forms is not distinguishable by steady state kinetic analysis. However, detailed EXAFS studies indicate that the isolated green recombinant enzyme initially contains a dioxo-molybdenum structure with no Mo-O bond, suggesting that its conversion to the brown protein with the native absorption spectrum involves loss of one of the oxo groups concomitant with the mooring of the molybdenum to Ser-147 ligand (41). This finding demonstrates a plasticity of the molybdenum-active site that may explain the structural variations previously observed.…”

Section: Discussionmentioning

confidence: 69%

Re-design of Rhodobacter sphaeroides Dimethyl Sulfoxide Reductase

Hilton

Temple

Rajagopalan

1999

Journal of Biological Chemistry

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The periplasmic DMSO reductase from Rhodobacter sphaeroides f. sp. denitrificans has been expressed in Escherichia coli BL21(DE3) cells in its mature form and with the R. sphaeroides or E. coli N-terminal signal sequence. Whereas the R. sphaeroides signal sequence prevents formation of active enzyme, addition of a 6؋ Histag at the N terminus of the mature peptide maximizes production of active enzyme and allows for affinity purification. The recombinant protein contains 1.7-1.9 guanines and greater than 0.7 molybdenum atoms per molecule and has a DMSO reductase activity of 3.4 -3.7 units/nmol molybdenum, compared with 3.7 units/nmol molybdenum for enzyme purified from R. sphaeroides. The recombinant enzyme differs from the native enzyme in its color and spectrum but is indistinguishable from the native protein after redox cycling with reduced methyl viologen and Me 2 SO. Substitution of Cys for the molybdenum-ligating Ser-147 produced a protein with DMSO reductase activity of 1.4 -1.5 units/nmol molybdenum. The mutant protein differs from wild type in its color and absorption spectrum in both the oxidized and reduced states. This substitution leads to losses of 61-99% of activity toward five substrates, but the adenosine N 1 -oxide reductase activity increases by over 400%.

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Section: Discussionmentioning

confidence: 69%

Re-design of Rhodobacter sphaeroides Dimethyl Sulfoxide Reductase

Hilton

Temple

Rajagopalan

1999

Journal of Biological Chemistry

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“…Dithionite reduction of BSO reductase produces a dramatic change in the resonance Raman spectrum in the Mo-S stretching region, but the intensity pattern and frequencies are similar to those of dithionite-reduced Me 2 SO reductase and can be assigned by direct analogy (Table I) (16,18,19) and is attributed to strengthening of the Mo-S bonds in response to loss of the oxo ligand.…”

Section: Resultsmentioning

confidence: 90%

“…Moreover, in contrast to the crystallographic data for this enzyme (2), both sets of molybdopterin dithiolate ligands were shown to remain firmly attached throughout the catalytic cycle (16). Active site structures analogous to those deduced from resonance Raman investigations were proposed independently for Me 2 SO reductase via molybdenum extended x-ray absorption fine structure (EAXFS) studies of the equivalent oxidized and reduced forms (18,19).…”

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confidence: 81%

“…There is also an exchangeable terminal oxygen ligand (oxo or hydroxo) that is lengthened (1.8 Å in the x-ray structure (5) and 1.9 Å by EXAFS (20)) compared with a typical terminal MoϭO group (1.7 Å) due to hydrogen bonding to a tryptophan residue, and it is this oxo group that constitutes the site of attack by Me 2 S (5,6,20). However, a catalytic scheme involving a spectator oxo group and a long terminal oxo group that is poised for oxygen atom transfer is not consistent with the resonance Raman and EXAFS data for R. sphaeroides Me 2 SO reductase (16,19). In addition to the absence of any evidence for a spectator oxo group in dithioniteor Me 2 S-reduced samples, the vibrational frequency of the substrate-and solvent-exchangeable oxo group can only be interpreted in terms of a short MoϭO (i.e.…”

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confidence: 98%

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Resonance Raman Characterization of Biotin Sulfoxide Reductase

Garton

Temple

Dhawan

et al. 2000

Journal of Biological Chemistry

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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 ...

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“…In the present case it is very similar to that in the spectra of oxidized active sites of enzymes of the sulfite oxidase family and of the CO dehydrogenase with only one molybdopterin ligand and two oxo ligands in a cis arrangement. [19][20][21][22][23] The experimental EXAFS (extended X-ray absorption fine structure) spectrum was initially fitted with the X-ray crystallography data 12 taking into account all six atoms directly coordinated to the molybdenum, the second molybdenum and the second sulfur (Fig. 3).…”

Section: Xas Spectroscopymentioning

confidence: 99%

The difference one ligand atom makes – An altered oxygen transfer reaction mechanism caused by an exchange of selenium for sulfur

Andrade

Meyer‐Klaucke

et al. 2010

Polyhedron

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Please cite this article as: C.E.A. Andrade, X. Ma, W. Meyer-Klaucke, C. Schulzke, The difference one ligand atom makes -an altered oxygen transfer reaction mechanism caused by an exchange of selenium for sulfur, Polyhedron (2009Polyhedron ( ), doi: 10.1016Polyhedron ( /j.poly.2009 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. ACCEPTED MANUSCRIPTThe difference one ligand atom makes -an altered oxygen transfer reaction mechanism caused by an exchange of selenium for sulfur Carlos Enrique Abad Andrade, [a] Xiaoli Ma, [b] Wolfram Meyer-Klaucke, [ AbstractThe influence of sulfur versus selenium coordination to molybdenum on the oxo transfer reaction mechanisms of functional models for oxidoreductases has been studied. The solution structure of the dimeric molybdenum compound with tridentate bis-anionic ligands containing a thioether function ( -O(CH 2 ) 3 S(CH 2 ) 3 O -) has been determined using EXAFS spectroscopy to be able to compare a feature of its solution structure to that of its selenoether analogue. A significant difference is found for the solution structures of the two compounds. The thioether group remains coordinated in solution, whereas the selenoether does not. The influence of this difference on the catalytic oxo transfer has been investigated in detail by following the catalytic transition of PPh 3 to OPPh 3 with DMSO as oxygen donor with variation of both substrate concentrations.

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Structure of the Molybdenum Site of Dimethyl Sulfoxide Reductase

Cited by 160 publications

References 60 publications

Re-design of Rhodobacter sphaeroides Dimethyl Sulfoxide Reductase

Re-design of Rhodobacter sphaeroides Dimethyl Sulfoxide Reductase

Resonance Raman Characterization of Biotin Sulfoxide Reductase

The difference one ligand atom makes – An altered oxygen transfer reaction mechanism caused by an exchange of selenium for sulfur

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