Infrared spectra of carbon monoxide complexes of indoleamine 2,3-dioxygenase and L-tryptophan 2,3-dioxygenases. Effects of substrates on the CO-stretching frequencies

Carbonmonoxy indoleamine 2,3-dioxygenase from rabbit small intestine exhibited two CO stretch bands at 1953 and 1933 cm-1 with half-band widths (delta v 1/2) of both approximately 15 cm-1. Upon addition of an excess amount of L-tryptophan, the substrate, the spectrum changed into that with an intense single band at 1902 cm-1 with the delta v 1/2 of 15 cm-1. Carbonmonoxy L-tryptophan 2,3-dioxygenase of Pseudomonas acidovorans in the absence of L-tryptophan showed a fused CO stretch band which consists of two components at 1965 and 1958 cm-1 (delta v 1/2 for the fused band; 25 cm-1), which was converted into a sharp single band at 1968 cm-1 (delta v 1/2; 10 cm-1) upon addition of excess L-tryptophan. On the other hand, CO complex of rat liver L-tryptophan 2,3-dioxygenase in the absence of L-tryptophan gave a spectrum with a poorly defined peak around 1961 cm-1. By the addition of L-tryptophan, the spectrum changed into that with two distinct bands at 1972 and 1920 cm-1 (delta v 1/2; 6 and 13 cm-1, respectively). These spectra were insensitive to pH in a range where the enzymes were not denatured (neutral to near pH 9). The infrared spectra of the carbonmonoxy enzymes were also affected by the addition of certain effectors such as skatole and alpha-methyl-DL-tryptophan, which facilitate the binding of L-tryptophan to the catalytic site of intestinal and Pseudomonas enzymes, respectively. However, the changes were of different types from those by the saturating amount of L-tryptophan. Possible mechanisms for these phenomena are discussed in relation to the structure of the heme-CO complex in these heme-containing dioxygenases.     

Rapid P450 heme iron reduction by laser photoexcitation of Mycobacterium tuberculosis CYP121 and CYP51B1. Analysis of CO complexation reactions and reversibility of the P450/P420 equilibrium

We demonstrate that photoexcitation of NAD(P)H reduces heme iron of Mycobacterium tuberculosis P450s CYP121 and CYP51B1 on the microsecond time scale. Rates of formation for the ferrous-carbonmonoxy (Fe(II)-CO) complex were determined across a range of coenzyme/CO concentrations. CYP121 reaction transients were biphasic. A hyperbolic dependence on CO concentration was observed, consistent with the presence of a CO binding site in ferric CYP121. CYP51B1 absorption transients for Fe(II)-CO complex formation were monophasic. The reaction rate was second order with respect to [CO], suggesting the absence of a CO-binding site in ferric CYP51B1. In the absence of CO, heme iron reduction by photoexcited NAD(P)H is fast ( approximately 10,000-11,000 s(-1)) with both P450s. For CYP121, transients revealed initial production of the thiolate-coordinated (P450) complex (absorbance maximum at 448 nm), followed by a slower phase reporting partial conversion to the thiol-coordinated P420 species (at 420 nm). The slow phase amplitude increased at lower pH values, consistent with heme cysteinate protonation underlying the transition. Thus, CO binding occurs to the thiolate-coordinated ferrous form prior to cysteinate protonation. For CYP51B1, slow conversions of both the ferrous/Fe(II)-CO forms to species with spectral maxima at 423/421.5 nm occurred following photoexcitation in the absence/presence of CO. This reflected conversion from ferrous thiolate- to thiol-coordinated forms in both cases, indicating instability of the thiolate-coordinated ferrous CYP51B1. CYP121 Fe(II)-CO complex pH titrations revealed reversible spectral transitions between P450 and P420 forms. Our data provide strong evidence for P420 formation linked to reversible heme thiolate protonation, and demonstrate key differences in heme chemistry and CO binding for CYP121 and CYP51B1.     

Effects of cholesterol analogues and inhibitors on the heme moiety of cytochrome P-450scc: a resonance Raman study

Interactions of cholesterol analogues and inhibitors with the heme moiety of cytochrome P-450scc were examined by resonance Raman spectroscopy. The Raman spectra of ferric cytochrome P-450scc complexed with inhibitors such as cyanide, phenyl isocyanide, aminoglutethimide, and metyrapone were characteristic of low-spin state and were very similar. However, the effect of exchange of the sixth ligand from the oxygen atom (ferric low-spin state) to the nitrogen atom upon aminoglutethimide and metyrapone binding was seen as down-frequency shifts of the v3 band from 1503 to 1501 and 1502 cm-1, respectively, while cyanide and phenyl isocyanide binding caused an up-frequency shift of the v3 band to 1505 cm-1. The effects of cholesterol analogues [22(R)-hydroxycholesterol, 22(S)-hydroxycholesterol, 22-ketocholesterol, 20(S)-hydroxycholesterol, and 25-hydroxycholesterol] on a Fe2+-CO stretching frequency of cytochrome P-450scc in ferrous CO form were examined. The 22(R)-hydroxycholesterol complex could not give a clear Fe2+-CO stretching Raman band due to a strong photodissociability. 22(S)-Hydroxycholesterol and 25-hydroxycholesterol complexes gave the Raman bands at 487 and 483 cm-1, respectively, whereas 20(S)-hydroxycholesterol and 22-ketocholesterol complexes gave Fe2+-CO stretching frequencies (478 cm-1) almost identical with that without substrate (477 cm-1). These findings suggest the existence of the following physiologically important natures of the cytochrome P-450scc active site: (1) there is a strong steric interaction between heme-bound carbon monoxide and the 22(R)-hydroxyl group or the 22(R)-hydrogen of the steroid side chain and (2) the hydroxylation at the 20S position may cause a conformational change of the side-chain group relative to the heme.     

Exchange reactions catalyzed by methioninase from Pseudomonas putida. Isolation and characterization of the exchange products.

Gel-electrophoretically homogeneous methioninase [L-methionine methanethiol-lyase (deaminating), EC 4.4.1.11] of Pseudomonas putida, which catalyzes alpha, beta- and alpha, gamma-eliminations from S-substituted amino acids, could also catalyze a variety of beta- and gamma-exchange reactions, according to the following equations: RSCH2CH(NH2)COOH+R'SH in equilibrium R'SCH2CH(NH2)COOH+RSH (beta-exchange) and RSCH2CH2CH(NH2)COOH+R'SH in equilibrium R'SCH2CH2CH(NH2)COOH+RSH (gamma-exchange), where R'SH represents an exogeneously added alkanethiol or a substituted thiol. Related amino acids not available for elimination reactions appeared to be inert as substrates for exchange. The maximum activity for the exchange reactions was observed at pH 8.5 in potassium pyrophosphate buffer. The activity increased linearly with the increase in protein concentration from zero to 3.0 mug per ml, and with incubation time up to at least 15 min at 30 degrees. Some of the exchange reaction products were purified by a combination of paper and ion exchange chromatographies, and charcoal treatment: their structures were confirmed by physicochemical methods including elemental analysis and proton magnetic resonance, infrared, and mass spectrometries.     

Mechanism of N-WASP activation by CDC42 and phosphatidylinositol 4, 5-bisphosphate

Neuronal Wiskott-Aldrich Syndrome protein (N-WASP) transmits signals from Cdc42 to the nucleation of actin filaments by Arp2/3 complex. Although full-length N-WASP is a weak activator of Arp2/3 complex, its activity can be enhanced by upstream regulators such as Cdc42 and PI(4,5)P(2). We dissected this activation reaction and found that the previously described physical interaction between the NH(2)-terminal domain and the COOH-terminal effector domain of N-WASP is a regulatory interaction because it can inhibit the actin nucleation activity of the effector domain by occluding the Arp2/3 binding site. This interaction between the NH(2)- and COOH termini must be intramolecular because in solution N-WASP is a monomer. Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) influences the activity of N-WASP through a conserved basic sequence element located near the Cdc42 binding site rather than through the WASp homology domain 1. Like Cdc42, PI(4,5)P(2) reduces the affinity between the NH(2)- and COOH termini of the molecule. The use of a mutant N-WASP molecule lacking this basic stretch allowed us to delineate a signaling pathway in Xenopus extracts leading from PI(4, 5)P(2) to actin nucleation through Cdc42, N-WASP, and Arp2/3 complex. In this pathway, PI(4,5)P(2) serves two functions: first, as an activator of N-WASP; and second, as an indirect activator of Cdc42.     

NMR studies of the activation of the Escherichia coli trp repressor

The Escherichia coli trp repressor binds to the trp operator in the presence of tryptophan, thereby inhibiting tryptophan biosynthesis. Tryptophan analogues lacking the alpha-amino group act as inducers of trp operon expression. We have used one- and two-dimensional 1H-NMR spectroscopy to compare the binding to the repressor of the corepressors L-tryptophan, D-tryptophan and 5-methyl-DL-tryptophan with that of the inducer indole-3-propionic acid. We have determined the chemical shifts of the indole ring protons of the ligands when bound to the protein, principally by magnetization-transfer experiments. The chemical shifts of the indole NH and C4 protons differ between corepressors and inducer. At the same time, the pattern of intermolecular NOE between protons of the protein and those of the ligand also differ between the two classes of ligand. These two lines of evidence indicate that corepressors and inducers bind differently in the binding site, and the evidence suggests that the orientation of the indole ring in the binding site differs by approximately 180 degrees between the two kinds of ligand. This is in contrast to a previous solution study [Lane, A.N. (1986) Eur. J. Biochem. 157, 405-413], but consistent with recent X-ray crystallographic work [Lawson, C.L. & Sigler, P.B. (1988) Nature 333, 869-871]. D-Tryptophan and 5-methyltryptophan, which are more effective corepressors than L-tryptophan, bind similarly to L-tryptophan. The indole ring of D-tryptophan appears to bind in essentially the same orientation as that of the L isomer. There are, however, some differences in chemical shifts and NOE for 5-methyltryptophan, which indicate that there are significant differences between the two corepressors L-tryptophan and 5-methyltryptophan in the orientation of the indole ring within the binding site.     

Allosteric effects acting over a distance of 20-25 A in the Escherichia coli tryptophan synthase bienzyme complex increase ligand affinity and cause redistribution of covalent intermediates

The reactions of L-histidine (L-His) and L-tryptophan (L-Trp) with the alpha 2 beta 2 complex of Escherichia coli tryptophan synthase are introduced as probes both of beta-subunit catalysis and of ligand-mediated alpha-beta allosteric interactions. Binding of DL-alpha-glycerol 3-phosphate (GP), an analogue of 3-indole-D-glycerol 3'-phosphate (IGP), to the alpha-catalytic site increases the affinity of alpha 2 beta 2 for L-His 4.5-fold and the affinity for L-Trp 17-fold and brings about a redistribution of beta-bound intermediates that favors the quinonoids derived from each amino acid. Inorganic phosphate (Pi) (presumably via binding to the alpha-catalytic site) influences the distribution of L-His intermediates as does GP. Previous binding studies [Heyn, M. P., & Weischet, W. O. (1975) Biochemistry 14, 2962-2968] indicate that when the phosphoryl group subsite of the alpha-catalytic site is occupied by GP or Pi, a high-affinity indole subsite is induced at the alpha-catalytic site. Interaction of benzimidazole (BZ), an analogue of indole, with this site also shifts the distribution of beta-bound L-His intermediates in favor of the L-His quinonoid. In the absence of Pi or GP, BZ interacts primarily at the beta-catalytic site and competes with L-His for the beta-subunit indole subsite. Since L-His and GP (or Pi) are substrate analogues and L-Trp is the physiological product, these allosteric effects likely take place with the natural substrates.(ABSTRACT TRUNCATED AT 250 WORDS)     

The tryptophan synthase bienzyme complex transfers indole between the alpha- and beta-sites via a 25-30 A long tunnel

The bacterial tryptophan synthase bienzyme complexes (with subunit composition alpha 2 beta 2) catalyze the last two steps in the biosynthesis of L-tryptophan. For L-tryptophan synthesis, indole, the common metabolite, must be transferred by some mechanism from the alpha-catalytic site to the beta-catalytic site. The X-ray structure of the Salmonella typhimurium tryptophan synthase shows the catalytic sites of each alpha-beta subunit pair are connected by a 25-30 A long tunnel [Hyde, C. C., Ahmed, S. A., Padlan, E. A., Miles, E. W., & Davies, D. R. (1988) J. Biol. Chem. 263, 17857-17871]. Since the S. typhimurium and Escherichia coli enzymes have nearly identical sequences, the E. coli enzyme must have a similar tunnel. Herein, rapid kinetic studies in combination with chemical probes that signal the bond formation step between indole (or nucleophilic indole analogues) and the alpha-aminoacrylate Schiff base intermediate, E(A-A), bound to the beta-site are used to investigate tunnel function in the E. coli enzyme. If the tunnel is the physical conduit for the transfer of indole from the alpha-site to the beta-site, then ligands that block the tunnel should also inhibit the rate at which indole and indole analogues from external solution react with E(A-A). We have found that when D,L-alpha-glycerol 3-phosphate (GP) is bound to the alpha-site, the rate of reaction of indole and nucleophilic indole analogues with E(A-A) is strongly inhibited. These compounds appear to gain access to the beta-site via the alpha-site and the tunnel, and this access is blocked by the binding of GP to the alpha-site. However, when small nucleophiles such as hydroxylamine, hydrazine, or N-methylhydroxylamine are substituted for indole, the rate of quinonoid formation is only slightly affected by the binding of GP. Furthermore, the reactions of L-serine and L-tryptophan with alpha 2 beta 2 show only small rate effects due to the binding of GP. From these experiments, we draw the following conclusions: (1) L-Serine and L-tryptophan gain access to the beta-site of alpha 2 beta 2 directly from solution. (2) The small effects of GP on the rates of the L-serine and L-tryptophan reactions are due to GP-mediated allosteric interactions between the alpha- and beta-sites.(ABSTRACT TRUNCATED AT 400 WORDS)     

Substitution of glutamic acid 109 by aspartic acid alters the substrate specificity and catalytic activity of the beta-subunit in the tryptophan synthase bienzyme complex from Salmonella typhimurium.

In an effort to understand the catalytic mechanism of the tryptophan synthase beta-subunit from Salmonella typhimurium, possible functional active site residues have been identified (on the basis of the 3-D crystal structure of the bienzyme complex) and targeted for analysis utilizing site-directed mutagenesis. The chromophoric properties of the pyridoxal 5'-phosphate cofactor provide a particularly convenient and sensitive spectral probe to directly investigate changes in catalytic events which occur upon modification of the beta-subunit. Substitution of Asp for Glu 109 in the beta-subunit was found to alter both the catalytic activity and the substrate specificity of the beta-reaction. Steady-state kinetic data reveal that the beta-reaction catalyzed by the beta E109D alpha 2 beta 2 mutant enzyme complex is reduced 27-fold compared to the wild-type enzyme. Rapid-scanning stopped-flow (RSSF) UV-visible spectroscopy shows that the mutation does not seriously affect the pre-steady-state reaction of the beta E109D mutant with L-serine to form the alpha-aminoacrylate intermediate, E(A-A). Binding of the alpha-subunit specific ligand, alpha-glycerol phosphate (GP) to the alpha 2 beta 2 complex exerts the same allosteric effects on the beta-subunit as observed with the wild-type enzyme. However, the pre-steady-state spectral changes for the reaction of indole with E(A-A) show that the formation of the L-tryptophan quinonoid, E(Q3), is drastically altered. Discrimination against E(Q3) formation is also observed for the binding of L-tryptophan to the mutant alpha 2 beta 2 complex in the reverse reaction. In contrast, substitution of Asp for Glu 109 increases the apparent affinity of the beta E109D alpha-aminoacrylate complex for the indole analogue indoline and results in the increased rate of synthesis of the amino acid product dihydroiso-L-tryptophan. Thus, the mutation affects the covalent bond forming addition reactions and the nucleophile specificity of the beta-reaction catalyzed by the bienzyme complex.     

Infrared evidence of cyanide binding to iron and copper sites in bovine heart cytochrome c oxidase. Implications regarding oxygen reduction

Cyanide binding to bovine heart cytochrome c oxidase at five redox levels has been investigated by use of infrared and visible-Soret spectra. A C-N stretch band permits identification of the metal ion to which the CN- is bound and the oxidation state of the metal. Non-intrinsic Cu, if present, is detected as a cyanide complex. Bands can be assigned to Cu+CN at 2093 cm-1, Cu2+CN at 2151 or 2165 cm-1, Fe3+CN at 2131 cm-1, and Fe2+CN at 2058 cm-1. Fe2+CN is found only when the enzyme is fully reduced whereas the reduced Cu+CN occurs in 2-, 3-, and 4-electron reduced species. A band for Fe3+CN is not found for the complex of fully oxidized enzyme but is for all partially reduced species. Cu2+CN occurs in both fully oxidized and 1-electron-reduced oxidase. CO displaces the CN- at Fe2+ to give a C-O band at 1963.5 cm-1 but does not displace the CN- at Cu+. Another metal site, noted by a band at 2042 cm-1, is accessible only in fully reduced enzyme and may represent Zn2+ or another Cu+. Binding of either CN- or CO may induce electron redistribution among metal centers. The extraordinary narrowness of ligand infrared bands indicates very little mobility of the components that line the O2 reduction site, a property of potential advantage for enzyme catalysis. The infrared evidence that CN- can bind to both Fe and Cu supports the possibility of an O2 reduction mechanism in which an intermediate with a mu-peroxo bridge between Fe and Cu is formed. On the other hand, the apparent independence of Fe and Cu ligand-binding sites makes a heme hydroperoxide (Fe-O-O-H) intermediate an attractive alternative to the formation an Fe-O-O-Cu linkage.     

Enzyme-induced strain/distortion in the ground-state ES complex in beta-lactamase catalysis revealed by FTIR

Class A beta-lactamases hydrolyze penicillins and other beta-lactams via an acyl-enzyme catalytic mechanism. Ser70 is the active site nucleophile. By constructing the S70A mutant, which is unable to form the acyl-enzyme intermediate, it was possible to make stable ES complexes with various substrates. The stability of such Michaelis complexes permitted acquisition of their infrared spectra. Comparison of the beta-lactam carbonyl stretch frequency (nu(CO)) in the free and enzyme-bound substrate revealed an average decrease of 13 cm(-)(1), indicating substantial strain/distortion of the lactam carbonyl when bound in the ES complex. Interestingly, regardless of the frequency of the C=O stretch in the free substrate, when complexed to Bacillus licheniformis beta-lactamase, the frequency was always 1755 +/- 2 cm(-)(1). This suggests the active site environment induces a similar conformation of the beta-lactam in all substrates when bound to the enzyme. Using deuterium substitution, it was shown that the "oxyanion hole", which involves hydrogen bonding to two backbone amides, is the major source of the enzyme-induced strain/distortion. The very weak catalytic activity of the S70A beta-lactamase suggests enzyme-facilitated hydrolysis due to substrate distortion on binding to the enzyme. Thus the binding of the substrate in the active site induces substantial strain and distortion that contribute significantly to the overall rate enhancement in beta-lactamase catalysis.     

Ligand, cofactor, and residue vibrations in the catalytic site of endothelial nitric oxide synthase

A study of bovine endothelial nitric oxide synthase by Fourier transform infrared (FTIR) spectroscopy in the 1000-2500 cm(-)(1) range is reported. Binding of CO to the reduced enzyme gives two heme(II)-CO nu(C)(-)(O) stretches (1927 and 1904 cm(-)(1)) which appear to be in rapid equilibrium. Photolysis of this heme(II)-CO compound is accompanied by perturbation of the local fine structure around the catalytic site giving vibrational changes of protein backbone, substrate, amino acid residues, and cofactors, to which heme, substrate arginine, and catalytic site residues contribute. Possible assignments of vibrations to heme, substrate arginine, and catalytic site residues are discussed. The discussion of assignments is informed by known structures, absorbance frequencies, and extinction coefficients of residues and cofactors, analysis of H(2)O-D(2)O exchange effects, analysis of substrate (14)N-(15)N (guanidinium)-arginine exchange effects, and comparison with the nNOS isoform (which differs in the replacement of asparagine 368 with an aspartate within the substrate binding site). The FTIR data can be modeled on the known structure of the catalytic site and indicate the extent of modulation of vibrational modes upon photolysis of the CO compound.     

Spectroscopic and equilibrium properties of the indoleamine 2,3-dioxygenase-tryptophan-O2 ternary complex and of analogous enzyme derivatives. Tryptophan binding to ferrous enzyme adducts with dioxygen, nitric oxide, and carbon monoxide

The dioxygen adduct of the heme protein indoleamine 2,3-dioxygenase has been generated at -30 degrees C in mixed solvents, and spectroscopic and equilibrium studies of its L-tryptophan (substrate) binding properties have been carried out for the first time. Comparative studies have also been performed with the NO and CO adducts of the ferrous enzyme. Under the conditions employed (-30 degrees C), both autoxidation and turnover (L-tryptophan + O2----formylkynurenine) of the ternary complex are effectively suppressed. Structural identification of the ternary complex is based on the 1:1 molar stoichiometry for the substrate-oxygenated enzyme adduct formation (Kd approximately 10(-4) M), the time-dependent linear product formation (turnover) at -20 degrees C, and the quantitative conversion of the complex to the ferrous CO derivative by bubbling with CO. Binding of L-tryptophan to the oxygenated enzyme leads to decreases in the intensities of its major absorption bands (lambda max 415, 541, 576 nm) and to a blue shift of its Soret peak. Interestingly, among the ferrous enzyme derivatives examined, only the substrate-bound oxygenated enzyme exhibits solvent-dependent Soret absorption peak positions, e.g., lambda max 411.5 and 413.5 nm in 65% (v/v) aqueous glycerol and ethylene glycol, respectively. In addition, indole binds to the oxygenated enzyme, causing a red shift of its Soret peak in these solvents only in the presence of substrate (411.5----414 nm and 413.5----414.5 nm, respectively), while similar effects of indole are independent of tryptophan for the other ferrous enzyme derivatives.(ABSTRACT TRUNCATED AT 250 WORDS)     

Crystal structures of CO and NO adducts of MauG in complex with pre-methylamine dehydrogenase: implications for the mechanism of dioxygen activation

MauG is a diheme enzyme responsible for the post-translational formation of the catalytic tryptophan tryptophylquinone (TTQ) cofactor in methylamine dehydrogenase (MADH). MauG can utilize hydrogen peroxide, or molecular oxygen and reducing equivalents, to complete this reaction via a catalytic bis-Fe(IV) intermediate. Crystal structures of diferrous, Fe(II)-CO, and Fe(II)-NO forms of MauG in complex with its preMADH substrate have been determined and compared to one another as well as to the structure of the resting diferric MauG-preMADH complex. CO and NO each bind exclusively to the 5-coordinate high-spin heme with no change in ligation of the 6-coordinate low-spin heme. These structures reveal likely roles for amino acid residues in the distal pocket of the high-spin heme in oxygen binding and activation. Glu113 is implicated in the protonation of heme-bound diatomic oxygen intermediates in promoting cleavage of the O-O bond. Pro107 is shown to change conformation on the binding of each ligand and may play a steric role in oxygen activation by positioning the distal oxygen near Glu113. Gln103 is in a position to provide a hydrogen bond to the Fe(IV)═O moiety that may account for the unusual stability of this species in MauG.     

The catalytic mechanism of tryptophan synthase from Escherichia coli. Kinetics of the reaction of indole with the enzyme--L-serine complexes

The mechanism by which indole condenses with L-serine in the active site of tryptophan synthase was studied by the stopped-flow technique. The single turnover occurs by rapid binding of indole to the pre-formed enzyme--L-serine complex, followed by C--C bond formation, reprotonation of the alpha carbon carbanion of L-tryptophan, and its final release. The effects of isotopic substitution at C-3 of indole, of pH, and of the presence of indolepropanol phosphate on these processes were also studied. The mechanism of binding of indole complements the known mechanisms of binding of L-serine and L-tryptophan to give a detailed picture of the mechanism of catalysis. It invokes two competent species of enzyme--L-serine complexes, leading to a branched pathway for the central condensation process. The rates of dehydration of L-serine and reprotonation of the carbanion of L-tryptophan are probably limited by rearrangements at the active site. Analysis of absorption, fluorescence and circular dichroic spectra, as well as of published data on the stereoisomers obtained by reduction with borohydride, suggests that the rearrangement includes a reorientation of the pyridoxal phosphate C-4' atom. The mechanism provides a detailed framework for explaining all available information, including the activating effect of the alpha subunit on the reaction catalyzed by the beta 2 subunit.     

Structural changes that occur upon photolysis of the Fe(II)(a3)-CO complex in the cytochrome ba(3)-oxidase of Thermus thermophilus: a combined X-ray crystallographic and infrared spectral study demonstrates CO binding to Cu(B)

The purpose of the work was to provide a crystallographic demonstration of the venerable idea that CO photolyzed from ferrous heme-a(3) moves to the nearby cuprous ion in the cytochrome c oxidases. Crystal structures of CO-bound cytochrome ba(3)-oxidase from Thermus thermophilus, determined at ~2.8-3.2? resolution, reveal a Fe-C distance of ~2.0?, a Cu-O distance of 2.4? and a Fe-C-O angle of ~126°. Upon photodissociation at 100K, X-ray structures indicate loss of Fe(a3)-CO and appearance of Cu(B)-CO having a Cu-C distance of ~1.9? and an O-Fe distance of ~2.3?. Absolute FTIR spectra recorded from single crystals of reduced ba(3)-CO that had not been exposed to X-ray radiation, showed several peaks around 1975cm(-1); after photolysis at 100K, the absolute FTIR spectra also showed a significant peak at 2050cm(-1). Analysis of the 'light' minus 'dark' difference spectra showed four very sharp CO stretching bands at 1970cm(-1), 1977cm(-1), 1981cm(-1), and 1985cm(-1), previously assigned to the Fe(a3)-CO complex, and a significantly broader CO stretching band centered at ~2050cm(-1), previously assigned to the CO stretching frequency of Cu(B) bound CO. As expected for light propagating along the tetragonal axis of the P4(3)2(1)2 space group, the single crystal spectra exhibit negligible dichroism. Absolute FTIR spectrometry of a CO-laden ba(3) crystal, exposed to an amount of X-ray radiation required to obtain structural data sets before FTIR characterization, showed a significant signal due to photogenerated CO(2) at 2337cm(-1) and one from traces of CO at 2133cm(-1); while bands associated with CO bound to either Fe(a3) or to Cu(B) in "light" minus "dark" FTIR difference spectra shifted and broadened in response to X-ray exposure. In spite of considerable radiation damage to the crystals, both X-ray analysis at 2.8 and 3.2? and FTIR spectra support the long-held position that photolysis of Fe(a3)-CO in cytochrome c oxidases leads to significant trapping of the CO on the Cu(B) atom; Fe(a3) and Cu(B) ligation, at the resolutions reported here, are otherwise unaltered.     

Characterization of the FAD-containing N-methyltryptophan oxidase from Escherichia coli

N-Methyltryptophan oxidase (MTOX) is a flavoenzyme that catalyzes the oxidative demethylation of N-methyl-L-tryptophan and other N-methyl amino acids, including sarcosine, which is a poor substrate. The Escherichia coli gene encoding MTOX (solA) was isolated on the basis of its sequence homology with monomeric sarcosine oxidase, a sarcosine-inducible enzyme found in many bacteria. These studies show that MTOX is expressed as a constitutive enzyme in a wild-type E. coli K-12 strain, providing the first evidence that solA is a functional gene. MTOX expression is enhanced 3-fold by growth on minimal media but not induced by N-methyl-L-tryptophan, L-tryptophan, or 3-indoleacrylate. MTOX forms an anionic flavin semiquinone and a reversible, covalent flavin-sulfite complex (K(d) = 1.7 mM), properties characteristic of flavoprotein oxidases. Rates of formation (k(on) = 5.4 x 10(-3) M(-1) s(-1)) and dissociation (k(off) = 1.3 x 10(-5) s(-1)) of the MTOX-sulfite complex are orders of magnitude slower than observed with most other flavoprotein oxidases. The pK(a) for ionization of oxidized FAD at N(3)H in MTOX (8.36) is two pH units lower than that observed for free FAD. The MTOX active site was probed by characterization of various substrate analogues that act as competitive inhibitors with respect to N-methyl-L-tryptophan. Qualitatively similar perturbations of the MTOX visible absorption spectrum are observed for complexes formed with various aromatic carboxylates, including benzoate, 3-indole-(CH(2))(n)-CO(2)(-) and 2-indole-CO(2)(-). The most stable complex with 3-indole-(CH(2))(n)-CO(2)(-) is formed with 3-indolepropionate (K(d) = 0.79 mM), a derivative with the same side chain length as N-methyl-L-tryptophan. Benzoate binding is enhanced upon protonation of a group in the enzyme-benzoate complex (pK(EL) = 6.87) but blocked by ionization of a group in the free enzyme (pK(E) = 8.41), which is attributed to N(3)H of FAD. Difference spectra observed for the aromatic carboxylate complexes are virtually mirror images of those observed with sarcosine analogues (N,N'-dimethylglycine, N-benzylglycine). Charge-transfer complexes are formed with 3-indoleacrylate, pyrrole-2-carboxylate, and CH(3)XCH(2)CO(2)(-) (X = S, Se, Te).     

Mass-spectrometric studies of the interrelations among hydrogenase, carbon monoxide dehydrogenase, and methane-forming activities in pure and mixed cultures of Desulfovibrio vulgaris, Desulfovibrio desulfuricans, and Methanosarcina barkeri

The activities of pure and mixed cultures of Desulfovibrio vulgaris and Methanosarcina barkeri in the exponential growth phase were monitored by measuring changes in dissolved-gas concentration by membrane-inlet mass spectrometry. M. barkeri grown under H2-CO2 or methanol produced limited amounts of methane and practically no hydrogen from either substrate. The addition of CO resulted in a transient H2 production concomitant with CO consumption. Hydrogen was then taken up, and CH4 production increased. All these events were suppressed by KCN, which inhibited carbon monoxide dehydrogenase activity. Therefore, with both substrates, H2 appeared to be an intermediate in CO reduction to CH4. The cells grown on H2-CO2 consumed 4 mol of CO and produced 1 mol of CH4. Methanol-grown cells reduced CH3OH with H2 resulting from carbon monoxide dehydrogenase activity, and the ratio was then 1 mol of CH4 to 1 mol of CO. Only 12CH4 and no 13CH4 was obtained from 13CO, indicating that CO could not be the direct precursor of CH4. In mixed cultures of D. vulgaris and M. barkeri on lactate, an initial burst of H2 was observed, followed by a lower level of production, whereas methane synthesis was linear with time. Addition of CO to the mixed culture also resulted in transient extra H2 production but had no inhibitory effect upon CH4 formation, even when the sulfate reducer was D. vulgaris Hildenborough, whose periplasmic iron hydrogenase is very sensitive to CO. The hydrogen transfer is therefore probably mediated by a less CO-sensitive nickel-iron hydrogenase from either of both species.     

Monomeric sarcosine oxidase: 1. Flavin reactivity and active site binding determinants

Monomeric sarcosine oxidase (MSOX) is an inducible bacterial flavoenzyme that catalyzes the oxidative demethylation of sarcosine (N-methylglycine) and contains covalently bound FAD [8alpha-(S-cysteinyl)FAD]. This paper describes the spectroscopic and thermodynamic properties of MSOX as well as the X-ray crystallographic characterization of three new enzyme.inhibitor complexes. MSOX stabilizes the anionic form of the oxidized flavin (pK(a) = 8.3 versus 10.4 with free FAD), forms a thermodynamically stable flavin radical, and stabilizes the anionic form of the radical (pK(a) < 6 versus pK(a) = 8.3 with free FAD). MSOX forms a covalent flavin.sulfite complex, but there appears to be a significant kinetic barrier against complex formation. Active site binding determinants were probed in thermodynamic studies with various substrate analogues whose binding was found to perturb the flavin absorption spectrum and inhibit MSOX activity. The carboxyl group of sarcosine is essential for binding since none is observed with simple amines. The amino group of sarcosine is not essential, but binding affinity depends on the nature of the substitution (CH(3)XCH(2)CO(2)(-), X = CH(2) < O < S < Se < Te), an effect which has been attributed to differences in the strength of donor-pi interactions. MSOX probably binds the zwitterionic form of sarcosine, as judged by the spectrally similar complexes formed with dimethylthioacetate [(CH(3))(2)S(+)CH(2)CO(2)(-)] and dimethylglycine (K(d) = 20.5 and 17.4 mM, respectively) and by the crystal structure of the latter. The methyl group of sarcosine is not essential but does contribute to binding affinity. The methyl group contribution varied from -3.79 to -0.65 kcal/mol with CH(3)XCH(2)CO(2)(-) depending on the nature of the heteroatom (NH(2)(+) > O > S) and appeared to be inversely correlated with heteroatom electron density. Charge-transfer complexes are formed with MSOX and CH(3)XCH(2)CO(2)(-) when X = S, Se, or Te. An excellent linear correlation is observed between the energy of the charge transfer bands and the one-electron reduction potentials of the ligands. The presence of a sulfur, selenium, or telurium atom identically positioned with respect to the flavin ring is confirmed by X-ray crystallography, although the increased atomic radius of S < Se < Te appears to simultaneously favor an alternate binding position for the heavier atoms. Although L-proline is a poor substrate, aromatic heterocyclic carboxylates containing a five-membered ring and various heteroatoms (X = NH, O, S) are good ligands (K(d, X=NH) = 1.37 mM) and form charge-transfer complexes with MSOX. The energy of the charge-transfer bands (S > O > NH) is linearly correlated with the one-electron ionization potentials of the corresponding heterocyclic rings.     

Mass-spectrometric studies of the interrelations among hydrogenase, carbon monoxide dehydrogenase, and methane-forming activities in pure and mixed cultures of Desulfovibrio vulgaris, Desulfovibrio desulfuricans, and Methanosarcina barkeri.

The activities of pure and mixed cultures of Desulfovibrio vulgaris and Methanosarcina barkeri in the exponential growth phase were monitored by measuring changes in dissolved-gas concentration by membrane-inlet mass spectrometry. M. barkeri grown under H2-CO2 or methanol produced limited amounts of methane and practically no hydrogen from either substrate. The addition of CO resulted in a transient H2 production concomitant with CO consumption. Hydrogen was then taken up, and CH4 production increased. All these events were suppressed by KCN, which inhibited carbon monoxide dehydrogenase activity. Therefore, with both substrates, H2 appeared to be an intermediate in CO reduction to CH4. The cells grown on H2-CO2 consumed 4 mol of CO and produced 1 mol of CH4. Methanol-grown cells reduced CH3OH with H2 resulting from carbon monoxide dehydrogenase activity, and the ratio was then 1 mol of CH4 to 1 mol of CO. Only 12CH4 and no 13CH4 was obtained from 13CO, indicating that CO could not be the direct precursor of CH4. In mixed cultures of D. vulgaris and M. barkeri on lactate, an initial burst of H2 was observed, followed by a lower level of production, whereas methane synthesis was linear with time. Addition of CO to the mixed culture also resulted in transient extra H2 production but had no inhibitory effect upon CH4 formation, even when the sulfate reducer was D. vulgaris Hildenborough, whose periplasmic iron hydrogenase is very sensitive to CO. The hydrogen transfer is therefore probably mediated by a less CO-sensitive nickel-iron hydrogenase from either of both species.