An adiabatic model for calculating overtone spectra of dimers such as (H(2)O)(2).

The near-infrared and visible wavelength spectrum of the water dimer is considered to be the major contributor to the so-called water continuum at these wavelengths. However, theoretical models of this spectrum require the simultaneous treatment of both monomer and dimer excitations. A model for treating this problem is proposed which is based upon a Franck-Condon-like separation between the monomer and dimer vibrational motions. In this model, one of the monomers is treated as the chromophore and its absorption is assumed to be given by its, possibly perturbed, vibrational band intensity. The main computational issue is the treatment of separate monomer and dimer motions. Various approaches for obtaining dimer vibration-rotation tunnelling spectra that allow for monomer motion are explored. These approaches include ways of treating the adiabatic separation of dimer vibrational modes from monomer vibrational modes. We classify the adiabatic separation methods under four main approaches: namely fixed-geometry, free-monomer, perturbed-monomer and coupled-monomer methods. The latter being the most computationally expensive as the monomer wave functions are dependent on the dimer coordinates. For each of these approaches, expectation values over the full potential are calculated for the given monomer vibrational wave functions. Various full (named VAP 2pD in the text) and partial (VAP (+p)D) averaging techniques are outlined to calculate the vibrationally averaged, monomer state-dependent, dimer interaction potentials. The computational costs associated with application of these techniques to the water dimer are estimated and the prospects for full calculations based on this approach are assessed.

Expression of a novel P275L variant of NADH:cytochrome b5 reductase gives functional insight into the conserved motif important for pyridine nucleotide binding.

The clinical disorder of recessive congenital methemoglobinemia (RCM, OMIN 250800) is associated with mutations in NADH:cytochrome b5 reductase (cb5r) and manifests as cyanosis from birth. Screening a cyanotic infant indicated elevated methemoglobin levels and decreased cb5r activity suggesting RCM. Sequencing the DIA1 gene encoding cb5r revealed a novel mutation, C27161T (NCBI accession number: NT_011520), resulting in replacement of proline at amino acid 275 with leucine (P275L). To understand how this mutation would affect cb5r's function, the P275L variant was expressed in a heterologous expression system and spectroscopic, thermodynamic, and thermostability studies were performed. The leucine substitution at residue 275 was found to significantly decrease the affinity towards the physiological reducing substrate, NADH, without affecting the activity of the P275L variant. From the rat model, residue 275 is predicted to be part of a conserved "CGPPPM" motif important for the binding and correct positioning of the NADH reducing substrate. Thus P275 influences the interaction with NADH which was confirmed by the change in affinity towards the physiological reducing substrate.

Identification and characterization of the novel FAD-binding lobe G75S mutation in cytochrome b(5) reductase: an aid to determine recessive congenital methemoglobinemia status in an infant.

NADH-cytochrome b(5) reductase deficiency results clinically in either type I or type II recessive congenital methemoglobinemia. The more severe type II form is associated with a global deficiency of cytochrome b(5) reductase and is characterized by cyanosis with neurological dysfunction. In contrast, the only symptom for type I is cyanosis. We have identified a novel G to A mutation at position 15,635 in the DIAI gene of a 4-month-old baby that results in a glycine to serine substitution at codon 75 in the cytochrome b(5) reductase protein. The G75S mutation, located in the FAD-binding lobe of cytochrome b(5) reductase, was found in association with the previously described V252M variant. The V252M mutation is present in the NADH-binding domain and associated with both types I and II recessive congenital methemoglobinemia. Since the G75S and V252M mutations represent radical changes in differing regions of cytochrome b(5) reductase, generating and characterizing these variants singly and in combination using a rat heterologous expression system would provide insight into the differences between types I and II disease at the molecular level. Although all three variants were found to retain stoichiometric levels of FAD with spectroscopic and thermodynamic properties comparable to those of native cytochrome b(5) reductase, all exhibited decreased catalytic efficiency and reduced protein stability reflecting the position of the mutations in the primary structure. The G75S variant retained only 11% of the catalytic efficiency of the wild-type enzyme. Thus, cytochrome b(5) reductase deficient patients who are heterozygous for either FAD- or NADH-binding lobe mutations can exhibit the clinically less severe type I phenotype.

[Ca2+] oscillations in a model of energy-dependent Ca2+ uptake by the endoplasmic reticulum.

Active Ca2+ transport in living cells necessitates controlled supply of metabolic energy. Direct coupling between sarco/endoplasmic reticulum (ER) Ca2+ ATPases (SERCA) and intracellular energy-generation sites has been well established experimentally. On the basis of these experimental findings we propose a pump-driven model to investigate complex dynamic properties of a cell system. The model describes the pump process both by the Ca2+ ATPase itself and by a suitable description of the glycolysis. The associated set of differential equations shows a rich behavior, the solutions ranging from simple periodic oscillations to complex patterns such as bursting and spiking. Recent experimental results on calcium oscillations in Xenopus laevis oocytes and on dynamic patterns of intracellular Ca2+ concentrations in electrically non-excitable cells are well described by corresponding theoretical results derived within the proposed model. The simulation results are further compared to spontaneous [Ca2+] oscillations in primitive endodermal cells.

Recessive congenital methaemoglobinaemia: functional characterization of the novel D239G mutation in the NADH-binding lobe of cytochrome b5 reductase.

Type I recessive congenital methaemoglobinaemia (RCM), caused by the reduced form of nicotinamide adenine dinucleotide (NADH)-cytochrome b(5) reductase (cytb(5)r) deficiency, manifests clinically as cyanosis without neurological dysfunction. Two mutations, E255- and G291D, have been identified in the NADH-binding lobe of cytb(5)r in previously reported patients, and we have detected a further novel mutation, D239G, in this lobe in two unrelated Irish families. Although one family belongs to the genetically isolated Traveller Community, which separated from the general Irish population during the 1845-48 famine, the D239G mutation was present on the same haplotype in both families. Three known cytb(5)r mutations were also identified, including the R159- mutation, which causes loss of the entire NADH-binding lobe and had previously been reported in an individual with type II RCM. Characterization of the three NADH-binding lobe mutants using a heterologous expression system revealed that all three variants retained stoichiometric levels of flavin adenine dinucleotide with spectroscopic and thermodynamic properties comparable with those of native cytb(5)r. In contrast to the E255- and G291D variants, the novel D239G mutation had no adverse impact on protein thermostability. The D239G mutation perturbed substrate binding, causing both decreased specificity for NADH and increased specificity for NADPH. Thus cytb(5)r deficient patients who are heterozygous for an NADH-binding lobe mutation can exhibit the clinically less severe type I phenotype, even in association with heterozygous deletion of the NADH-binding lobe.

A genome-screen of a large twin cohort reveals linkage for quantitative ultrasound of the calcaneus to 2q33-37 and 4q12-21.

UNLABELLED: A genome-wide screen was performed on a large cohort of dizygous twin pairs to identify regions of the genome that contain QTL for QUS of bone. Suggestive linkage of QUS parameters to 2q33-37 and 4q12-21 highlighted these regions as potentially important for studies of genes that regulate bone. INTRODUCTION: The genetics of osteoporotic fracture is only partly explained by bone mineral density (BMD). Quantitative ultrasound (QUS) of the calcaneus can also be used for independent clinical assessment of osteoporotic fracture risk. Two specific indices are derived from this assessment: broadband ultrasound attenuation (BUA) and velocity of sound (VOS). Both parameters provide information on fracture risk; however, BUA has been studied more extensively and may be favored because it is thought to have a stronger predictive value for osteoporotic fracture and incorporates aspects of trabecular structure and bone quality as well as BMD. Studies of QUS in twins have shown that both derived parameters are under substantial genetic control, independent of BMD. MATERIALS AND METHODS: To identify regions of the genome that contain quantitative trait loci (QTL) for QUS of bone, we performed a genome-wide screen on a large cohort of dizygous twin pairs. Unselected female dizygous twins from 1067 pedigrees from the St Thomas' UK Adult Twin Registry were genome scanned (737 highly polymorphic microsatellite markers). Multipoint linkage analyses provided maximum evidence of linkage for BUA (LOD 2.1-5.1) to 2q33-37. Linkage for VOS (LOD 2.2-3.4) was maximal at 4q12-21. Potential evidence of linkage in the cohort indicated five other possible locations of QTL (LOD > 2.0) relevant to bone density or structure on chromosomes 1, 2, 13, 14, and X. RESULTS AND CONCLUSIONS: This study has identified eight genomic locations with linkage of LOD > 2.0. This data should be of value in assisting researchers to localize genes that regulate bone mass and microstructure. These results should complement genome screens of BMD and bone structure and serve to enable further targeted positional candidate and positional cloning studies to advance our understanding of genetic control of bone quality and risk of fracture.

Generating neural circuits that implement probabilistic reasoning.

We extend the hypothesis that neuronal populations represent and process analog variables in terms of probability density functions (PDFs). Aided by an intermediate representation of the probability density based on orthogonal functions spanning an underlying low-dimensional function space, it is shown how neural circuits may be generated from Bayesian belief networks. The ideas and the formalism of this PDF approach are illustrated and tested with several elementary examples, and in particular through a problem in which model-driven top-down information flow influences the processing of bottom-up sensory input.

Neural representation of probabilistic information.

It has been proposed that populations of neurons process information in terms of probability density functions (PDFs) of analog variables. Such analog variables range, for example, from target luminance and depth on the sensory interface to eye position and joint angles on the motor output side. The requirement that analog variables must be processed leads inevitably to a probabilistic description, while the limited precision and lifetime of the neuronal processing units lead naturally to a population representation of information. We show how a time-dependent probability density rho(x; t) over variable x, residing in a specified function space of dimension D, may be decoded from the neuronal activities in a population as a linear combination of certain decoding functions phi(i)(x), with coefficients given by the N firing rates a(i)(t) (generally with D << N). We show how the neuronal encoding process may be described by projecting a set of complementary encoding functions phi;(i)(x) on the probability density rho(x; t), and passing the result through a rectifying nonlinear activation function. We show how both encoders phi;(i)(x) and decoders phi(i)(x) may be determined by minimizing cost functions that quantify the inaccuracy of the representation. Expressing a given computation in terms of manipulation and transformation of probabilities, we show how this representation leads to a neural circuit that can carry out the required computation within a consistent Bayesian framework, with the synaptic weights being explicitly generated in terms of encoders, decoders, conditional probabilities, and priors.

The structure and biochemistry of NADH-dependent cytochrome b5 reductase are now consistent.

Cytochrome b5 reductase (cb5r) (EC 1.6.6.2) catalyzes the reduction of two molecules of cytochrome b5 using NADH as the physiological electron donor. The structure of pig cb5r at 2.4 A resolution was previously reported in the literature, but it was inconsistent with the biochemistry; for example, K83 and C245 were both implicated in the mechanism, but were not located at the active site. To address this problem, we have determined the structures of cb5r from rat at 2.0 A resolution and in a complex with NAD+ at 2.3 A resolution. We found significant differences throughout the rat structure compared to that of pig, including the locations of the lysine and cysteine residues mentioned above. To test the structural models, we made single amino acid substitutions of this lysine and showed that all substitutions produced correctly folded proteins and exhibited normal flavin behavior. However, the apparent kcat(NADH) decreased, and the apparent K(m) for NADH increased; the K(m)'s for cytochrome b5 were unchanged relative to that of the wild type. The largest effect was for the glutamate-substituted protein, which was further characterized using a charge transfer assay and found to be less efficient at NADH utilization than the wild type. These results are consistent with a role for this lysine in stabilizing the NADH-bound form of cb5r. We have concluded that the pig structure was mistraced in several regions and have reinterpreted mutants in these regions that give rise to the hereditary disease methemoglobinemia.

Production of a recombinant hybrid hemoflavoprotein: engineering a functional NADH:cytochrome c reductase.

A gene has been constructed coding for a unique fusion protein, NADH:cytochrome c reductase, that comprises the soluble heme-containing domain of rat hepatic cytochrome b(5) as the amino-terminal portion of the protein and the soluble flavin-containing domain of rat hepatic cytochrome b(5) reductase as the carboxyl terminus. The gene has been expressed in Escherichia coli resulting in the highly efficient production of a functional hybrid hemoflavoprotein which has been purified to homogeneity by a combination of ammonium sulfate precipitation, affinity chromatography on 5'-ADP agarose, and size-exclusion chromatography. The purified protein exhibited a molecular mass of approximately 46 kDa by polyacrylamide gel electrophoresis and 40,875 Da, for the apoprotein, using mass spectrometry which also confirmed the presence of both heme and FAD prosthetic groups. The fusion protein showed immunological cross-reactivity with both anti-rat cytochrome b(5) and anti-rat cytochrome b(5) reductase antibodies indicating the conservation of antigenic determinants from both native domains. Spectroscopic analysis indicated the fusion protein contained both a b-type cytochrome and flavin chromophors with properties identical to those of the native proteins. Amino-terminal and internal amino acid sequencing confirmed the identity of peptides derived from both the heme- and flavin-binding domains with sequences identical to the deduced amino acid sequence. The isolated fusion protein retained NADH:ferricyanide reductase activity (k(cat) = 8.00 x 10(2) s(-1), K(NADH)(m) = 4 microM, K(FeCN(6))(m) = 11 microM) comparable to that of that of native NADH:cytochrome b(5) reductase and also exhibited both NADH:cytochrome c reductase activity (k(cat) = 2.17 x 10(2) s(-1), K(NADH)(m) = 2 microM, K(FeCN(6))(m) = 11 microM, K(Cyt.c)(m) = 1 microM) and NADH:methemoglobin reductase activity (k(cat) = 4.40 x 10(-1) s(-1), K(NADH)(m) = 3 microM, K(mHb)(m) = 47 microM), the latter two activities indicating efficient electron transfer from FAD to heme and retention of physiological function. This work represents the first successful bacterial expression of a soluble, catalytically competent, rat hepatic cytochrome b(5)-cytochrome b(5) reductase fusion protein that retains the functional properties characteristic of the individual heme and flavin domain.

Assimilatory nitrate reductase: lysine 741 participates in pyridine nucleotide binding via charge complementarity.

Assimilatory NADH:nitrate reductase (EC 1.6.6.1), a complex Mo-pterin-, cytochrome b557-, and FAD-containing protein, catalyzes the regulated and rate-limiting step in the utilization of inorganic nitrogen by high plants. With a recombinant, histidine-tagged form of the spinach nitrate reductase flavin domain, site-directed mutagenesis has been utilized to examine the role of lysine 741 in binding the reducing substrate, NADH. Seven individual mutants, corresponding to K741R, K741H, K741A, K741E, K741M, K741Q, and K741P, have been engineered and six of the resulting proteins purified to homogeneity. With the exception of K741P, all the mutants were obtained as functional flavoproteins which retained FAD as the sole prosthetic group and exhibited spectroscopic properties comparable to those of the wild-type domain, indicating that the amino acid substitutions had no effect on FAD binding. In contrast, all the mutants were found to have altered NADH:ferricyanide reductase (NADH:FR) activity with mutations affecting both kcat and K(NADH)m, which decreased and increased, respectively. At pH 7.0, kcat decreased in the order WT > K741R > K741A > K741H > K741E > K741M > K741Q while K(NADH)m increased in the same order. The most efficient mutant, K741R, retained 80% of the wild-type NADH:FR activity, while in contrast the most inefficient mutant, K741Q, retained only 18% of the wild-type NADH:FR activity together with a 118-fold increased K(NADH)m. pH studies of K741H revealed that both kcat and K(NADH)m were pH-dependent, with enhanced activity observed at acidic pH. These results indicated that retention of a positively charged side chain at position 741 in the spinach nitrate reductase primary sequence is important for the efficient binding and subsequent oxidation of NADH and that the positively charged side chain enhances nucleotide binding via charge complementarity with the negatively charged pyrophosphate moiety.

Arginine 91 is not essential for flavin incorporation in hepatic cytochrome b(5) reductase.

Cytochrome b(5) reductase (cb5r) catalyzes the transfer of reducing equivalents from NADH to cytochrome b(5). Utilizing an efficient heterologous expression system that produces a histidine-tagged form of the hydrophilic, diaphorase domain of the enzyme, site-directed mutagenesis has been used to generate cb5r mutants with substitutions at position 91 in the primary sequence. Arginine 91 is an important residue in binding the FAD prosthetic group and part of a conserved "RxY(T)(S)xx(S)(N)" sequence motif that is omnipresent in the "ferredoxin:NADP(+) reductase" family of flavoproteins. Arginine 91 was replaced with K, L, A, P, D, Q, and H residues, respectively, and all the mutant proteins purified to homogeneity. Individual mutants were expressed with variable efficiency and all exhibited molecular masses of approximately 32 kDa. With the exception of R91H, all the mutants retained visible absorption spectra typical of a flavoprotein, the former being produced as an apoprotein. Visible absorption spectra of R91A, L, and P were red shifted with maxima at 458 nm, while CD spectra indicated an altered FAD environment for all the mutants except R91K. Fluorescence spectra showed a reduced degree of intrinsic flavin fluorescence quenching for the R91K, A, and P, mutants, while thermal stability studies suggested all the mutants, except R91K, were somewhat less stable than the wild-type domain. Initial-rate kinetic measurements demonstrated that the mutants exhibited decreased NADH:ferricyanide reductase activity with the R91P mutant retaining the lowest activity, corresponding to a k(cat) of 283 s(-1) and a K(NADH)(m) of 105 microM, when compared to the wild-type domain (k(cat) = 800 s(-1) K(NADH)(m) = 6 microM). These results demonstrate that R91 is not essential for FAD binding in cb5r; however, mutation of R91 perturbs the flavin environment and alters both diaphorase substrate recognition and utilization.

Substitution of murine ferrochelatase glutamate-287 with glutamine or alanine leads to porphyrin substrate-bound variants.

Ferrochelatase (EC 4.99.1.1) is the terminal enzyme of the haem biosynthetic pathway and catalyses iron chelation into the protoporphyrin IX ring. Glutamate-287 (E287) of murine mature ferrochelatase is a conserved residue in all known sequences of ferrochelatase, is present at the active site of the enzyme, as inferred from the Bacillus subtilis ferrochelatase three-dimensional structure, and is critical for enzyme activity. Substitution of E287 with either glutamine (Q) or alanine (A) yielded variants with lower enzymic activity than that of the wild-type ferrochelatase and with different absorption spectra from the wild-type enzyme. In contrast to the wild-type enzyme, the absorption spectra of the variants indicate that these enzymes, as purified, contain protoporphyrin IX. Identification and quantification of the porphyrin bound to the E287-directed variants indicate that approx. 80% of the total porphyrin corresponds to protoporphyrin IX. Significantly, rapid stopped-flow experiments of the E287A and E287Q variants demonstrate that reaction with Zn(2+) results in the formation of bound Zn-protoporphyrin IX, indicating that the endogenously bound protoporphyrin IX can be used as a substrate. Taken together, these findings suggest that the structural strain imposed by ferrochelatase on the porphyrin substrate as a critical step in the enzyme catalytic mechanism is also accomplished by the E287A and E287Q variants, but without the release of the product. Thus E287 in murine ferrochelatase appears to be critical for the catalytic process by controlling the release of the product.

Circular permutation of 5-aminolevulinate synthase. Mapping the polypeptide chain to its function.

5-Aminolevulinate synthase is the first enzyme of the heme biosynthetic pathway in non-plant eukaryotes and some prokaryotes. The enzyme functions as a homodimer and requires pyridoxal 5'-phosphate as a cofactor. Although the roles of defined amino acids in the active site and catalytic mechanism have been recently explored using site-directed mutagenesis, much less is known about the role of the 5-aminolevulinate synthase polypeptide chain arrangement in folding, structure, and ultimately, function. To assess the importance of the continuity of the polypeptide chain, circularly permuted 5-aminolevulinate synthase variants were constructed through either rational design or screening of an engineered random library. One percent of the random library clones were active, and a total of 21 active variants had sequences different from that of the wild type 5-aminolevulinate synthase. Out of these 21 variants, 9 displayed unique circular permutations of the 5-aminolevulinate synthase polypeptide chain. The new termini of the active variants disrupted secondary structure elements and loop regions and fell in 100 amino acid regions from each terminus. This indicates that the natural continuity of the 5-aminolevulinate synthase polypeptide chain and the sequential arrangement of the secondary structure elements are not requirements for proper folding, binding of the cofactor, or assembly of the two subunits. Furthermore, the order of two identified functional elements (i.e. the catalytic and the glycine-binding domains) is apparently irrelevant for proper functioning of the enzyme. Although the wild type 5-aminolevulinate synthase and the circularly permuted variants appear to have similar, predicted overall tertiary structures, they exhibit differences in the arrangement of the secondary structure elements and in the cofactor-binding site environment. Taken together, the data lead us to propose that the 5-aminolevulinate synthase overall structure can be reached through multiple or alternative folding pathways.

Kinetic and mechanistic properties of biotin sulfoxide reductase.

Rhodobacter sphaeroides f. sp. denitrificans biotin sulfoxide reductase catalyzes the reduction of d-biotin d-sulfoxide (BSO) to biotin. Initial rate studies of the homogeneous recombinant enzyme, expressed in Escherichia coli, have demonstrated that the purified protein utilizes NADPH as a facile electron donor in the absence of any additional auxiliary proteins. We have previously shown [Pollock, V. V., and Barber, M. J. (1997) J. Biol. Chem. 272, 3355-3362] that, at pH 8 and in the presence of saturating concentrations of BSO, the enzyme exhibits, a marked preference for NADPH (k(cat,app) = 500 s(-1), K(m,app) = 269 microM, and k(cat,app)/K(m,app) = 1.86 x 10(6) M(-1) s(-1)) compared to NADH (k(cat,app) = 47 s(-1), K(m,app) = 394 microM, and k(cat,app)/K(m,app) = 1.19 x 10(5) M(-1) s(-1)). Production of biotin using NADPH as the electron donor was confirmed by both the disk biological assay and by reversed-phase HPLC analysis of the reaction products. The purified enzyme also utilized ferricyanide as an artificial electron acceptor, which effectively suppressed biotin sulfoxide reduction and biotin formation. Analysis of the enzyme isolated from tungsten-grown cells yielded decreased reduced methyl viologen:BSO reductase, NADPH:BSO reductase, and NADPH:FR activities, confirming that Mo is required for all activities. Kinetic analyses of substrate inhibition profiles revealed that the enzyme followed a Ping Pong Bi-Bi mechanism with both NADPH and BSO exhibiting double competitive substrate inhibition. Replots of the 1/v-axes intercepts of the parallel asymptotes obtained at several low concentrations of fixed substrate yielded a K(m) for BSO of 714 and 65 microM for NADPH. In contrast, utilizing NADH as an electron donor, the replots yielded a K(m) for BSO of 132 microM and 1.25 mM for NADH. Slope replots of data obtained at high concentrations of BSO yielded a K(i) for BSO of 6.10 mM and 900 microM for NADPH. Kinetic isotope studies utilizing stereospecifically deuterated NADPD indicated that BSO reductase uses specifically the 4R-hydrogen of the nicotinamide ring. Cyanide inhibited NADPH:BSO and NADPH:FR activities in a reversible manner while diethylpyrocarbonate treatment resulted in complete irreversible inactivation of the enzyme concomitant with molybdenum cofactor release, indicating that histidine residues are involved in cofactor-binding.

Bid-induced cytochrome c release is mediated by a pathway independent of mitochondrial permeability transition pore and Bax.

Bid, a pro-apoptosis "BH3-only" member of the Bcl-2 family, can be cleaved by caspase-8 after Fas/TNF-R1 engagement. The p15 form of truncated Bid (tBid) translocates to mitochondria and induces cytochrome c release, leading to the activation of downstream caspases and apoptosis. In the current study, we investigated the mechanism by which tBid regulated cytochrome c release in terms of its relationship to mitochondrial permeability transition and Bax, another Bcl-2 family protein. We employed an in vitro reconstitution system as well as cell cultures and an animal model to reflect the physiological environment where Bid could be functional. We found that induction of cytochrome c release by tBid was not accompanied by a permeability transition even at high doses. Indeed, inhibition of permeability transition did not suppress the activity of tBid in vitro nor could they block Fas activation-induced, Bid-dependent hepatocyte apoptosis in cultures. Furthermore, Mg(2+), although inhibiting permeability transition, actually enhanced the ability of tBid to induce cytochrome c release. We also found that tBid did not require Bax to induce cytochrome c release in vitro. In addition, mice deficient in bax were still highly susceptible to anti-Fas-induced hepatocyte apoptosis, in which cytochrome c release was unaffected. Moreover, although Bax-induced cytochrome c release was not dependent on tBid, the two proteins could function synergistically. We conclude that Bid possesses the biochemical activity to induce cytochrome c release through a mechanism independent of mitochondrial permeability transition pore and Bax.

Serine 121 is an essential amino acid for biotin sulfoxide reductase functionality.

Rhodobacter sphaeroides f. sp. denitrificans biotin sulfoxide reductase (BSOR) catalyzes the reduction of d-biotin d-sulfoxide (BSO) to biotin, an important step in oxidized vitamin salvaging. In addition to BSO, the enzyme also catalyzes the reduction of a variety of other substrates, including methionine sulfoxide, with decreased efficiencies, suggesting a potential role as a general cell protector against oxidative damage. Recombinant BSOR, expressed as a glutathione S-transferase fusion protein, contains the molybdopterin guanine dinucleotide cofactor (MGD) as its sole prosthetic group, which is required for the reduction of BSO by either NADPH or reduced methyl viologen. Comparison of the amino acid sequences of BSOR and the closely related MGD-containing enzyme, dimethyl sulfoxide reductase, has indicated a number of conserved residues, including an active site serine residue, serine 121, which has been potentially identified as the fifth coordinating ligand of Mo in BSOR. Site-directed mutagenesis has been used to replace serine 121 with cysteine, threonine, or alanine residues in the BSOR sequence to asses the role of this residue in catalysis and/or Mo coordination. All three BSOR mutant proteins were expressed, purified to homogeneity, and demonstrated to contain both MGD by fluorescence spectroscopy and Mo by inductively coupled plasma mass spectrometry, similar to wild-type enzyme. However, all three mutant proteins were devoid of BSOR activity using either NADPH or reduced methyl viologen as the electron donor. These results strongly suggest that serine 121 in BSOR is essential for catalysis but is not essential for either Mo coordination or MGD binding.

Structure of the molybdenum site of Rhodobacter sphaeroides biotin sulfoxide reductase.

Conditions for heterologous expression of Rhodobacter sphaeroides biotin sulfoxide reductase in Escherichia coli were modified, resulting in a significant improvement in the yield of recombinant enzyme and enabling structural studies of the molybdenum center. Quantitation of the guanine and the molybdenum as compared to that found in R. sphaeroides DMSO reductase demonstrated the presence of the bis(MGD)molybdenum cofactor. UV-visible absorption spectra were obtained for the oxidized, NADPH-reduced, and dithionite-reduced enzyme. EPR spectra were obtained for the Mo(V) state of the enzyme. X-ray absorption spectroscopy at the molybdenum K-edge has been used to probe the molybdenum coordination of the enzyme. The molybdenum site of the oxidized protein possesses a Mo(VI) mono-oxo site (Mo=O at 1.70 A) with additional coordination by approximately four thiolate ligands at 2.41 A and probably one oxygen or nitrogen at 1.95 A. The NADPH- and dithionite-reduced Mo(IV) forms of the enzyme are des-oxo molybdenum sites with approximately four thiolates at 2.33 A and two different Mo-O/N ligands at 2.19 and 1.94 A.

Resonance Raman characterization of biotin sulfoxide reductase. Comparing oxomolybdenum enzymes in the ME(2)SO reductase family.

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)O/(16)O labeling studies involving water and the alternative substrate dimethyl sulfoxide and the close correspondence to the resonance Raman spectra previously reported for dimethyl sulfoxide reductase (Garton, S. D., Hilton, J., Oku, H., Crouse, B. R., Rajagopalan, K. V., and Johnson, M. K. (1997) J. Am. Chem. Soc. 119, 12906-12916), vibrational modes associated with a terminal oxo ligand and the two molybdopterin dithiolene ligands have been assigned. The results indicate that the enzyme cycles between mono-oxo-Mo(VI) and des-oxo-Mo(IV) forms with both molybdopterin dithiolene ligands remaining coordinated in both redox states. Direct evidence for an oxygen atom transfer mechanism is provided by (18)O/(16)O labeling studies, which show that the terminal oxo group at the molybdenum center is exchangeable with water during redox cycling and originates from the substrate in substrate-oxidized samples. Biotin sulfoxide reductase is not reduced by biotin or the nonphysiological products, dimethyl sulfide and trimethylamine. However, product-induced changes in the Mo=O stretching frequency provide direct evidence for a product-associated mono-oxo-Mo(VI) catalytic intermediate. The results indicate that biotin sulfoxide reductase is thermodynamically tuned to catalyze the reductase reaction, and a detailed catalytic mechanism is proposed.

The pro-apoptotic proteins, Bid and Bax, cause a limited permeabilization of the mitochondrial outer membrane that is enhanced by cytosol.

During apoptosis, an important pathway leading to caspase activation involves the release of cytochrome c from the intermembrane space of mitochondria. Using a cell-free system based on Xenopus egg extracts, we examined changes in the outer mitochondrial membrane accompanying cytochrome c efflux. The pro-apoptotic proteins, Bid and Bax, as well as factors present in Xenopus egg cytosol, each induced cytochrome c release when incubated with isolated mitochondria. These factors caused a permeabilization of the outer membrane that allowed the corelease of multiple intermembrane space proteins: cytochrome c, adenylate kinase and sulfite oxidase. The efflux process is thus nonspecific. None of the cytochrome c-releasing factors caused detectable mitochondrial swelling, arguing that matrix swelling is not required for outer membrane permeability in this system. Bid and Bax caused complete release of cytochrome c but only a limited permeabilization of the outer membrane, as measured by the accessibility of inner membrane-associated respiratory complexes III and IV to exogenously added cytochrome c. However, outer membrane permeability was strikingly increased by a macromolecular cytosolic factor, termed PEF (permeability enhancing factor). We hypothesize that PEF activity could help determine whether cells can recover from mitochondrial cytochrome c release.