Mechanism of reaction of melatonin with human myeloperoxidase

Recently, it was suggested that melatonin (N-acetyl-5-methoxytryptamine) is oxidized by activated neutrophils in a reaction most probably involving myeloperoxidase (Biochem. Biophys. Res. Commun. (2000) 279, 657-662). Myeloperoxidase (MPO) is the most abundant protein of neutrophils and is involved in killing invading pathogens. To clarify if melatonin is a substrate of MPO, we investigated the oxidation of melatonin by its redox intermediates compounds I and II using transient-state spectral and kinetic measurements at 25 degrees C. Spectral and kinetic analysis revealed that both compound I and compound II oxidize melatonin via one-electron processes. The second-order rate constant measured for compound I reduction at pH 7 and pH 5 are (6.1 +/- 0.2) x 10(6) M(-1) s(-1) and (1.0 +/- 0.08) x 10(7) M(-1) s(-1), respectively. The rates for the one-electron reduction of compound II back to the ferric enzyme are (9.6 +/- 0.3) x 10(2) M(-1) s(-1) (pH 7) and (2.2 +/- 0.1) x 10(3) M(-1) s(-1) (pH 5). Thus, melatonin is a much better electron donor for compound I than for compound II. Steady-state experiments showed that the rate of oxidation of melatonin is dependent on the H(2)O(2) concentration, is not affected by superoxide dismutase, and is quickly terminated by sodium cyanide. Melatonin can markedly inhibit the chlorinating activity of MPO at both pH 7 and pH 5. The implication of these findings in the activated neutrophil is discussed.     

Mechanism of reaction of myeloperoxidase with nitrite

Myeloperoxidase (MPO) is a major neutrophil protein and may be involved in the nitration of tyrosine residues observed in a wide range of inflammatory diseases that involve neutrophils and macrophage activation. In order to clarify if nitrite could be a physiological substrate of myeloperoxidase, we investigated the reactions of the ferric enzyme and its redox intermediates, compound I and compound II, with nitrite under pre-steady state conditions by using sequential mixing stopped-flow analysis in the pH range 4-8. At 15 degrees C the rate of formation of the low spin MPO-nitrite complex is (2.5 +/- 0.2) x 10(4) m(-1) s(-1) at pH 7 and (2.2 +/- 0.7) x 10(6) m(-1) s(-1) at pH 5. The dissociation constant of nitrite bound to the native enzyme is 2.3 +/- 0.1 mm at pH 7 and 31.3 +/- 0.5 micrometer at pH 5. Nitrite is oxidized by two one-electron steps in the MPO peroxidase cycle. The second-order rate constant of reduction of compound I to compound II at 15 degrees C is (2.0 +/- 0.2) x 10(6) m(-1) s(-1) at pH 7 and (1.1 +/- 0.2) x 10(7) m(-1) s(-1) at pH 5. The rate constant of reduction of compound II to the ferric native enzyme at 15 degrees C is (5.5 +/- 0.1) x 10(2) m(-1) s(-1) at pH 7 and (8.9 +/- 1.6) x 10(4) m(-1) s(-1) at pH 5. pH dependence studies suggest that both complex formation between the ferric enzyme and nitrite and nitrite oxidation by compounds I and II are controlled by a residue with a pK(a) of (4.3 +/- 0.3). Protonation of this group (which is most likely the distal histidine) is necessary for optimum nitrite binding and oxidation.     

Characterization of binding of leukotriene C4 by human multidrug resistance protein 1: evidence of differential interactions with NH2- and COOH-proximal halves of the protein

Multidrug resistance protein 1 (MRP1) is capable of actively transporting a wide range of conjugated and unconjugated organic anions. The protein can also transport additional conjugated and unconjugated compounds in a GSH- or S-methyl GSH-stimulated manner. How MRP1 binds and transports such structurally diverse substrates is not known. We have used [(3)H]leukotriene C(4) (LTC(4)), a high affinity glutathione-conjugated physiological substrate, to photolabel intact MRP1, as well as fragments of the protein expressed in insect cells. These studies revealed that: (i) LTC(4) labels sites in the NH(2)- and COOH-proximal halves of MRP1, (ii) labeling of the NH(2)-half of MRP1 is localized to a region encompassing membrane-spanning domain (MSD) 2 and nucleotide binding domain (NBD) 1, (iii) labeling of this region is dependent on the presence of all or part of the cytoplasmic loop (CL3) linking MSD1 and MSD2, but not on the presence of MSD1, (iv) labeling of the NH(2)-proximal site is preferentially inhibited by S-methyl GSH, (v) labeling of the COOH-proximal half of the protein occurs in a region encompassing transmembrane helices 14-17 and appears not to require NBD2 or the cytoplasmic COOH-terminal region of the protein, (vi) labeling of intact MRP1 by LTC(4) is strongly attenuated in the presence of ATP and vanadate, and this decrease in labeling is attributable to a marked reduction in LTC(4) binding to the NH(2)-proximal site, and (vii) the attenuation of LTC(4) binding to the NH(2)-proximal site is a consequence of ATP hydrolysis and trapping of Vi-ADP exclusively at NBD2. These data suggest that MRP1-mediated transport involves a conformational change, driven by ATP hydrolysis at NBD2, that alters the affinity with which LTC(4) binds to one of two sites composed, at least in part, of elements in the NH(2)-proximal half of the protein.     

Re-evaluation of the allometry of wet thermal conductance for birds

Wet thermal conductance is an important thermoregulatory parameter for birds and mammals. It is generally calculated as C(wet) (ml O2 g(-1) h(-1) degrees C(-1)) = VO2/(T(b)-T(a)), where VO2 is metabolic rate measured in ml O2 g(-1) h(-1), T(b) is body and T(a) is ambient temperature measured in degrees C. Minimum C(wet) is measured at T(a) at or below the lower critical temperature (T(lc)) of the thermoneutral zone, and is strongly influenced by time of day (rest or activity phase) and body mass [J. Aschoff, Comp. Biochem. Physiol. 69A (1981) 611]. Allometric analyses indicate differences in C(wet) for passerine and non-passerine birds, in their rest and active phases (Aschoff, 1981). The allometric slope for non-passerine rest-phase (-0.583) is lower than that for non-passerine active-phase (-0.484), and passerine rest-phase (-0.461) and active-phase (-0.463), although none of these slopes are significantly different. This different-sloped relationship for non-passerine rest-phase C(wet) extrapolates to lower-than-expected values at high body mass, and so this allometric relationship may be inappropriate for predictive purposes. Consequently, we have reanalysed Aschoff's (1981) data, as well as more recent compilations, to determine a more useful allometric relationship for C(wet) of non-passerine rest-phase birds. Re-analyses of minimum thermal conductance data from Drent and Stonehouse [Comp. Biochem. Physiol. 40A (1971) 689], Aschoff (1981) and Gavrilov and Dolnik [Acta XVIII Congressus Internationalis Ornithologici Moscow (1982) 421] indicate that the most appropriate regressions for predicting C(wet) (ml O2 g(-1) h(-1) degrees C(-1)) of birds from body mass (M; g) are the pooled regressions for non-passerine and passerine birds, in the active (alpha) and resting (rho) phases, using data tabulated by Aschoff (1981): alpha, C(wet)=0.994M(-0.509); rho, C(wet)=0.702M(-0.519). C(wet) is approximately 40% higher in the active phase than the rest phase. Regressions of various data sets for C(wet) of birds and mammals indicate a similar slope of approximately -0.5 for the allometric relationship, but significantly higher elevations for mammals compared to birds. The approximately 50% higher C(wet) for mammals than birds indicates a better physical insulation for birds than mammals of the same body mass. The general scaling of C(wet) with M(-0.5) indicates that (T(b)-T(lc)) should scale with M(0.22), if mass-specific metabolic rate scales with M(-0.28) [Reynolds and Lee, Am. Nat. 147 (1996) 735]. The observed scaling for (T(b)-T(lc)) of M(0.183) (calculated from Gavrilov and Dolnik, 1985) is consistent with this expectation.     

Inhibition of pyrophosphatase activity of mouse duodenal alkaline phosphatase by magnesium ions

Duodenal alkaline phosphatase of juvenile (11-day-old) mice, like other non-specific alkaline phosphatases, has the ability to hydrolyse PP(i). When a constant Mg(2+)/PP(i) concentration ratio is maintained, plots of velocity as a function of PP(i) concentration are consistent with Michaelis-Menten kinetics. Mg(2+) activates pyrophosphate hydrolysis and maximal activity is obtained at a constant Mg(2+)/PP(i) concentration ratio of 0.66. At higher ratios there is strong inhibition. At constant concentrations of Mg(2+) and increasing concentrations of PP(i), the velocity-substrate (PP(i)) concentration plots show sigmoidal dependence. By assuming that the true substrate is MgP(2)O(7) (2-) complex, and using complexity constants, the concentrations of free Mg(2+), Mg(2)P(2)O(7) and MgP(2)O(7) (2-) were calculated in assay mixtures ranging in PP(i) concentration from 0.1 to 2.5mm and in total Mg(2+) concentration from 0.6 to 2.6mm. From these data, the concentrations of added Mg(2+) and PP(i) in the assay mixtures were selected so that the velocity could be measured (1) at three fixed concentrations of free Mg(2+) ions with varied concentrations of MgP(2)O(7) (2-) and (2) at four fixed concentrations of Mg(2)P(2)O(7) with varied concentrations of MgP(2)O(7) (2-). Lineweaver-Burk and Hill plots from these data showed that the inhibition is caused by free Mg(2+) ions, of a mixed type and consistent with Michaelis-Menten kinetics. The sigmoidal dependence observed between velocity and PP(i) concentration at constant concentration of total Mg(2+) is therefore not due to allosteric inhibition. It is due to a combined effect of (1) inhibition by free Mg(2+) ions, (2) depletion of the true substrate, MgP(2)O(7) (2-), owing to the formation of Mg(2)P(2)O(7) and (3) the manner in which the concentrations of these three molecular or ionic species change when PP(i) concentration is increased maintaining the total Mg(2+) concentration constant.     

The origin of the genetical diversity of Microtus mandarinus chromosomes

Here we describe our comparative studies on two types of X chromosomes, namely X(M) and X(SM,) of the mandarin vole (Microtus mandarinus). By chromosome G- and C-banding analysis, we have found that two different types of X chromosomes exist in mandarin voles. The two types of X chromosomes present two different G- and C-banding patterns: the X(M) chromosome is a longer metacentric X chromosome which is C-band negative; and the X(SM) is a shorter submetacentric X chromosome which has one C-band at the centromere and another one at the middle part of the short arm. The X(SM) has 6 G-bands including one on the kinetochore, one in the middle of the short arm, and four on the long arm. The X(M) has 7 G-bands including one on the kinetochore, two on the short arm, and four on the long arm. We have further found that female voles can be grouped into three types based on the composition of the X chromosome but the male voles have only one type. The three female groups are: (1) female voles (X(M)X(SM)), in which the two X chromosomes are different, the longer one is metacentric and the shorter is submetacentric; (2) female vole (X(SM)X(SM)), in which the two X chromosomes are both submetacentric; (3) female vole (X(M)O), in which there is only one X chromosome that is metacentric. Surprisingly, we have never found female voles with X(M)X(M), females with X(SM)O or males with X(M)Y. We hypothesize that the X(SM) chromosome is derived from the X(M) through its breakage and re-joining. The paper also discusses the formation of X(M)O females.     

Identification of functionally important residues of Arabidopsis thaliana S-adenosylmethionine decarboxylase

The Arabidopsis thaliana S-adenosylmethionine decarboxylase (AdoMetDC) cDNA (GenBank(TM) U63633) was cloned, and the AdoMetDC protein was expressed, purified, and characterized. The K(m) value for S-adenosylmethionine (AdoMet) is 23.1 microM and the K(i) value for methylglyoxal bis-(guanylhydrazone) (MGBG) is 0.15 microM. Site-specific mutagenesis was performed on the AdoMetDC to introduce mutations at conserved cysteine (Cys(50), Cys(83), and Cys(230)) and lysine(81) residues, chosen by examination of the conserved sequence and proved to be involved in enzymatic activity by chemical modification. The AdoMetDC mutants K81A and C83A retained up to 60 and 10% of wild type activity, respectively, demonstrating that lysyl and sulfhydryl groups are required for full catalytic activity. However, changing Cys(50) and Cys(230) to alanine had minimal effects on the catalytic activity. Changing Lys(81) to alanine produced an altered substrate specificity. When lysine was used as a substrate instead of AdoMet, the substrate specificity for lysine increased 6-fold. The K(m) value for AdoMet is 11-fold higher than that of the wild type, but the V(max) value is more than 60%. Taken together, the results suggest that the lysine(81) residue is critical for substrate binding.     

The antioxidant functions of cytochrome c

Low (C(1/2) = 1.5 x 10(-7) M) concentrations of horse cytochrome c strongly inhibit H(2)O(2) production by rat heart mitochondria under conditions of reverse electron transfer from succinate to NAD(+). The effect is abolished by binding of cytochrome c with liposomes and is not prevented by SOD. Yeast cytochrome c is much less effective than the horse protein whereas acetylated horse cytochrome c is without effect. H(2)O(2) formation stimulated by antimycin A is resistant to added cytochrome c. In inside-out submitochondrial vesicles, H(2)O(2) production is suppressed by all three cytochrome c samples tested, but at higher concentrations (C(1/2) is about 5 x 10(-7) M). In vesicles, SOD abolishes the cytochrome c inhibition. We conclude that extramitochondrial cytochrome c is competent in down-regulation of the Complex I H(2)O(2) production linked to the reverse electron transfer. Such an effect is absent in the inside-out submitochondrial vesicles where another antioxidant cytochrome c function can be observed, i.e. the oxidation of O(2-*) to O(2). A possible role of cytochrome c in the antioxidant defence is discussed.     

Interaction of isozymes of myosin subfragment 1 with actin: effect of ionic strength and nucleotide

Myosin subfragment 1 (S-1) can be fractionated into two isozymes, (A1)S-1 containing alkali light chain 1 and (A2)S-1 containing alkali light chain 2. The predominant difference in the behavior of the two isozymes of S-1 is that, at low ionic strength, the actin concentration required for half-maximal ATPase activity is considerably lower for (A1)S-1 than for (A2)S-1; that is, the apparent binding constant KATPase for (A1)S-1 is greater than KATPase for (A2)S-1 [Weeds, A.G., & Taylor, R.S. (1975) Nature (London) 257, 54-56]. This difference disappears at high ionic strength [Wagner, P. D., Slater, C. S., Pope, B., & Weeds, A.G. (1979) Eur. J. Biochem. 99, 385-394]. In the present study we investigated whether the difference in the KATPase values of (A1)S-1 and (A2)S-1 is due to a difference in the actual affinity of these S-1 isozymes for actin. Binding was measured in the presence of ATP and AMP-PNP and in the absence of nucleotide at varied ionic strengths. We found that at low ionic strength where KATPase is several times stronger for (A1)S-1 than for (A2)S-1, the binding of (A1)S-1 to actin is correspondingly stronger than that of (A2)S-1 irrespective of the nucleotide present. Furthermore, as the ionic strength is increased, just as the difference between the KATPase values for (A1)S-1 and (A2)S-1 disappears so too does the difference in the affinity of the two isozymes for actin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Roles of protein kinase C and actin-binding protein 280 in the regulation of intracellular trafficking of dopamine D3 receptor

D(3) dopamine receptor (D(3)R) is expressed mainly in parts of the brain that control the emotional behaviors. It is believed that the improper regulation of D(3)R is involved in the etiology of schizophrenia. Desensitization of D(3)R is weakly associated with G protein-coupled receptor kinase (GRK)/beta-arrestin-directed internalization. This suggests that there might be an alternative pathway that regulates D(3)R signaling. This report shows that D(3)R undergoes robust protein kinase C (PKC)-dependent sequestration that is accompanied by receptor phosphorylation and the desensitization of signaling. PKC-dependent D(3)R sequestration, which was enhanced by PKC-beta or -delta, was dynamin dependent but independent of GRK, beta-arrestin, or caveolin 1. Site-directed mutagenesis of all possible phosphorylation sites within the intracellular loops of D(3)R identified serine residues at positions 229 and 257 as the critical amino acids responsible for phorbol-12-myristate-13-acetate (PMA)-induced D(3)R phosphorylation, sequestration, and desensitization. In addition, the LxxY endocytosis motif, which is located between residues 252 and 255, was found to play accommodating roles for PMA-induced D(3)R sequestration. A continuous interaction with the actin-binding protein 280 (filamin A), which was previously known to interact with D(3)R, is required for PMA-induced D(3)R sequestration. In conclusion, the PKC-dependent but GRK-/beta-arrestin-independent phosphorylation of D(3)R is the main pathway responsible for the sequestration and desensitization of D(3)R. Filamin A is essential for both the efficient signaling and sequestration of D(3)R.     

Studies on the mechanism of alkaline peroxide delignification of agricultural residues

Alkaline solutions of hydrogen peroxide partially delignify wheat straw and other lignocellulosic materials, leaving a cellulosic residue that is highly susceptible to enzymatic digestion by cellulase. The delignification reaction is strongly dependent upon the pH of the reaction mixture, with an optimum at pH 11.5-11.6, pKa for the dissociation H(2)O(2) right harpoon over left harpoon H(+) + HOO(-). The data are consistent with a mechanism in which H(2)O(2) decomposition products such as .OH and O(2) (-)., rather than H(2)O(2) or HOO(-), are the primary lignin oxidizing species. During the course of the delignification reaction, O(2) is evolved from the reaction mixture indicating active H(2)O(2) decomposition. At a given concentration of H(2)O(2), the rate of O(2) evolution is proportional to the amount of lignocellulosic substrate present in the reaction mixture. However, the total amount of O(2) evolved is inversely proportional to the amount of substrate present, indicating that some of the peroxide oxygen becomes incorporated into lignin degradation products. The amount of peroxide oxygen incorporated can range as high as 2 O(2) per lignin C(9) unit, depending upon the initial concentration of lignocellulosic substrate.     

Effect of glycosylation on the function of a soluble, recombinant form of the transferrin receptor

Production of the soluble portion of the transferrin receptor (sTFR) by baby hamster kidney (BHK) cells is described, and the effect of glycosylation on the biological function of sTFR is evaluated for the first time. The sTFR (residues 121-760) has three N-linked glycosylation sites (Asn251, Asn317, and Asn727). Although fully glycosylated sTFR is secreted into the tissue culture medium ( approximately 40 mg/L), no nonglycosylated sTFR could be produced, suggesting that carbohydrate is critical to the folding, stability, and/or secretion of the receptor. Mutants in which glycosylation at positions 251 and 727 (N251D and N727D) is eliminated are well expressed, whereas production of the N317D mutant is poor. Analysis by electrospray ionization mass spectrometry confirms dimerization of the sTFR and the absence of the carbohydrate at the single site in each mutant. The effect of glycosylation on binding to diferric human transferrin (Fe(2) hTF), an authentic monoferric hTF with iron in the C-lobe (designated Fe(C) hTF), and a mutant (designated Mut-Fe(C) hTF that features a 30-fold slower iron release rate) was determined by surface plasmon resonance; a small ( approximately 20%) but consistent difference is noted for the binding of Fe(C) hTF and the Mut-Fe(C) hTF to the sTFR N317D mutant. The rate of iron release from Fe(C) hTF and Mut-Fe(C) hTF in complex with the sTFR and the sTFR mutants at pH 5.6 reveals that only the N317D mutant has a significant effect. The carbohydrate at position 317 lies close to a region of the TFR previously shown to interact with hTF.     

Structure and function of RNA replicase of bacteriophage Qbeta.

(1) The RNA replicase induced by bacteriophage Qbeta consists of four non-identical subunits designated as alpha (mol. wt. 74000), beta (mol. wt. 64000), gamma (mol. wt. 47000) and delta (mol. wt. 33000), only one (subunit beta) of which is specified by the phage genome. (2) Subunit alpha (30 S ribosomal protein "S1" as well as translational interference factor "i") is required only for (+) strand-directed RNA synthesis in the presence of the host factor. (3) Qbeta replicase lacking subunit alpha (R-alpha) is capable of replicating templates other than (+) strand, such as (--), "6S" RNA, poly(C) etc., in the absence of the host factor. (4) Subunit beta is suggested to be the nucleotide-polymerizing enzyme, but is unable to initiate RNA synthesis by itself. (5) Subunits gamma and delta are identical to the protein synthesis elongation factors, EF-Tu and EF-Ts, respectively, and are required only for initiation of RNA synthesis, but not for elongation. (6) A model of Qbeta replicase is presented in order to discuss observed template-enzyme interactions.     

Stereochemical specificity of organophosphorus acid anhydrolase toward p-nitrophenyl analogs of soman and sarin

Organophosphorus acid anhydrolase (OPAA) catalyzes the hydrolysis of p-nitrophenyl analogs of the organophosphonate nerve agents, sarin and soman. The enzyme is stereoselective toward the chiral phosphorus center by displaying a preference for the R(P)-configuration of these analogs. OPAA also exhibits an additional preference for the stereochemical configuration at the chiral carbon center of the soman analog. The preferred configuration of the chiral carbon center is dependent upon the configuration at the phosphorus center. The enzyme displays a two- to four-fold preference for the R(P)-enantiomer of the sarin analog. The k(cat)/K(m) of the R(P)-enantiomer is 250 M(-1) s(-1), while that of the S(P)-enantiomer is 110 M(-1) s(-1). The order of preference for the stereoisomers of the soman analog is R(P)S(C) > R(P)R(C) > S(P)R(C) > S(P)S(C). The k(cat)/K(m) values are 36,300 M(-1)s(-1), 1250 M(-1) s(-1), 80 M(-1) s(-1) and 5 M(-1) s(-1), respectively. The R(P)S(C)-isomer of the soman analog is therefore preferred by a factor of 7000 over the S(P)S(C)-isomer.     

Dimer formation of subunit G of the yeast V-ATPase

The G subunit of the vacuolar ATPase (V-ATPase) is a component of the stalk connecting the V(1) and V(O) sectors of the enzyme and is essential for normal assembly and function. Subunit G (Vma10p) of the yeast V-ATPase was expressed in Escherichia coli as a soluble protein and was purified to homogeneity. The molecular mass of subunit G, determined by Native-polyacrylamide gel electrophoresis, gel filtration analysis and small-angle X-ray scattering, was approximately 28+/-2 kDa, indicating that this protein is dimeric. With a radius of gyration (R(g)) and a maximum size (D(max)) of 2.7+/-0.2 nm and 8.0+/-0.3 nm, respectively, the G-dimer is rather elongated. To understand which region of subunit G is required to mediate dimerization, a G(38-144) form (the carboxyl-terminus) was expressed and purified. G(38-144) is homogeneous, with a molecular mass of approximately 12+/-3 kDa, indicating a monomeric form in solution.     

Photofootprinting of DNA triplexes.

We have used a photofootprinting assay to study intermolecular and intramolecular DNA triplexes. The assay is based on the fact that the DNA duplex is protected against photodamage (specifically, against the formation of the (6-4) pyrimidine photoproducts) within a triplex structure. We have shown that this is the case for PyPuPu (YRR) as well as PyPuPy (YRY) triplexes. Using the photofootprinting assay, we have studied the triplex formation under a variety of experimentally defined conditions. At acid pH, d(C)n.d(G)n.d(C)n and d(CT)n.d(GA)n.d(CT)n triplexes are detected by this method. The d(CT)n.d(GA)n.d(CT)n triplexes are additionally stabilized by divalent cations and spermidine. PyPuPu triplexes are pH-independent and are stabilized by divalent cations, such as Mg++ and Zn++. The effect depends on the type of cation and on the DNA sequence. The d(CT)n.d(GA)n.d(GA)n triplex is stabilized by Zn++, but not by Mg++, whereas the d(C)n.d(G)n.d(G)n triplex is stabilized by Mg++. In H-DNA, virtually the entire pyrimidine chain is protected against photodimerization, whereas only half of the pyrimidine chain participating in a triplex is protected in the CGG intramolecular triplex.     

Reactions of dimethylsulfoxide reductase in the presence of dimethyl sulfide and the structure of the dimethyl sulfide-modified enzyme

The bis-molybdopterin enzyme dimethylsulfoxide reductase (DMSOR) from Rhodobacter capsulatus catalyzes the conversion of dimethyl sulfoxide (DMSO) to dimethyl sulfide (DMS), reversibly, in the presence of suitable e(-)-donors or e(-)-acceptors. The catalytically significant intermediate formed by reaction of DMSOR with DMS ('the DMS species') and a damaged enzyme form derived by reaction of the latter with O(2) (DMS-modified enzyme, DMSOR(mod)D) have been investigated. Evidence is presented that Mo in the DMS species is not, as widely assumed, Mo(IV). Formation of the DMS species is reversed on removing DMS or by addition of an excess of DMSO. Equilibrium constants for the competing reactions of DMS and DMSO with the oxidized enzyme (K(d) = 0.07 +/- 0.01 and 21 +/- 5 mM, respectively) that control these processes indicate formation of the DMS species occurs at a redox potential that is 80 mV higher than that required, according to the literature, for reduction of Mo(VI) to Mo(IV) in the free enzyme. Specificity studies show that with dimethyl selenide, DMSOR yields a species analogous to the DMS species but with the 550 nm peak blue-shifted by 27 nm. It is concluded from published redox potential data that this band is due to metal-to-ligand charge transfer from Mo(V) to the chalcogenide. Since the DMS species gives no EPR signal in the normal or parallel mode, a free radical is presumed to be in close proximity to the metal, most likely on the S. The species is thus formulated as Mo(V)-O-S(*)Me(2). Existing X-ray crystallographic and Raman data are consistent with this structure. Furthermore, 1e(-) oxidation of the DMS species with phenazine ethosulfate yields a Mo(V) form without an -OH ligand, since its EPR signal shows no proton splittings. This form presumably arises via dissociation of DMSO. The structure of DMSOR(mod)D has been determined by X-ray crystallography. All four thiolate ligands and Ogamma of serine-147 remain coordinated to Mo, but there are no terminal oxygen ligands and Mo is Mo(VI). Thus, it is a dead-end species, neither oxo group acceptance nor e(-)-donation being possible. O(2)-dependent formation of DMSOR(mod)D represents noncatalytic breakdown of the DMS species by a pathway alternative to that in turnover, with oxidation to Mo(VI) presumably preceding product release. Steps in the forward and backward catalytic cycles are discussed in relation to earlier stopped-flow data. The finding that in the back-assay the Mo(IV) state may at least in part be by-passed via two successive 1e(-) reactions of the DMS species with the e(-)-acceptor, may have implications in relation to the existence of separate molybdopterin enzymes catalyzing DMSO reduction and DMS oxidation, respectively.