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R Hille

Publications and source records attributed to R Hille.

At least 37 records · Page 2Linked to original sources
PubMed1999

Redox cycles in trimethylamine dehydrogenase and mechanism of substrate inhibition.

The steady-state reaction of trimethylamine dehydrogenase (TMADH) with the artificial electron acceptor ferricenium hexafluorophosphate (Fc(+)) has been studied by stopped-flow spectroscopy, with particular reference to the mechanism of inhibition by trimethylamine (TMA). Previous studies have suggested that the presence of alternate redox cycles is responsible for the inhibition of activity seen in the high-substrate regime. Here, we demonstrate that partitioning between these redox cycles (termed the 0/2 and 1/3 cycles on the basis of the number of reducing equivalents present in the oxidized/reduced enzyme encountered in each cycle) is dependent on both TMA and electron acceptor concentration. The use of Fc(+) as electron acceptor has enabled a study of the major redox forms of TMADH present during steady-state turnover at different concentrations of substrate. Reduction of Fc(+) is found to occur via the 4Fe-4S center of TMADH and not the 6-S-cysteinyl flavin mononucleotide: the direction of electron flow is thus analogous to the route of electron transfer to the physiological electron acceptor, an electron-transferring flavoprotein (ETF). In steady-state reactions with Fc(+) as electron acceptor, partitioning between the 0/2 and 1/3 redox cycles is dependent on the concentration of the electron acceptor. In the high-concentration regime, inhibition is less pronounced, consistent with the predicted effects on the proposed branching kinetic scheme. Photodiode array analysis of the absorption spectrum of TMADH during steady-state turnover at high TMA concentrations reveals that one-electron reduced TMADH-possessing the anionic flavin semiquinone-is the predominant species. Conversely, at low concentrations of TMA, the enzyme is predominantly in the oxidized form during steady-state turnover. The data, together with evidence derived from enzyme-monitored turnover experiments performed at different concentrations of TMA, establish the operation of the branched kinetic scheme in steady-state reactions. With dimethylbutylamine (DMButA) as substrate, the partitioning between the 0/2 and 1/3 redox cycles is poised more toward the 0/2 cycle at all DMButA concentrations studied-an observation that is consistent with the inability of DMButA to act as an effective inhibitor of TMADH.

Diethylamines↗
PubMed1999

Rubredoxin from the green sulfur bacterium Chlorobium tepidum functions as an electron acceptor for pyruvate ferredoxin oxidoreductase.

Rubredoxin (Rd) from the moderately thermophilic green sulfur bacterium Chlorobium tepidum was found to function as an electron acceptor for pyruvate ferredoxin oxidoreductase (PFOR). This enzyme, which catalyzes the conversion of pyruvate to acetyl-CoA and CO(2), exhibited an absolute dependence upon the presence of Rd. However, Rd was incapable of participating in the pyruvate synthase or CO(2) fixation reaction of C. tepidum PFOR, for which two different reduced ferredoxins are employed as electron donors. These results suggest a specific functional role for Rd in pyruvate oxidation and provide the initial indication that the two important physiological reactions catalyzed by PFOR/pyruvate synthase are dependent on different electron carriers in the cell. The UV-visible spectrum of oxidized Rd, with a monomer molecular weight of 6500, gave a molar absorption coefficient at 492 nm of 6.89 mM(-1) cm(-1) with an A(492)/A(280) ratio of 0.343 and contained one iron atom/molecule. Further spectroscopic studies indicated that the CD spectrum of oxidized C. tepidum Rd exhibited a unique absorption maximum at 385 nm and a shoulder at 420 nm. The EPR spectrum of oxidized Rd also exhibited unusual anisotropic resonances at g = 9.675 and g = 4.322, which is composed of a narrow central feature with broader shoulders to high and low field. The midpoint reduction potential of C. tepidum Rd was determined to be -87 mV, which is the most electronegative value reported for Rd from any source.

Chlorobi↗
PubMed1999

Reductive half-reaction of the H172Q mutant of trimethylamine dehydrogenase: evidence against a carbanion mechanism and assignment of kinetically influential ionizations in the enzyme-substrate complex.

The reactions of wild-type trimethylamine dehydrogenase (TMADH) and of a His-172-->Gln (H172Q) mutant were studied by rapid-mixing stopped-flow spectroscopy over the pH range 6.0-10.5, to address the potential role of His-172 in abstracting a proton from the substrate in a 'carbanion' mechanism for C-H bond cleavage. The pH-dependence of the limiting rate for flavin reduction (klim) was studied as a function of pH for the wild-type enzyme with perdeuterated trimethylamine as substrate. The use of perdeuterated trimethylamine facilitated the unequivocal identification of two kinetically influential ionizations in the enzyme-substrate complex, with macroscopic pKa values of 6.5+/-0.2 and 8.4+/-0.1. A plot of klim/Kd revealed a bell-shaped curve and two kinetically influential ionizations with macroscopic pKa values of 9.4+/-0.1 and 10.5+/-0.1. Mutagenesis of His-172, a potential active-site base and a component of a novel Tyr-His-Asp triad in the active site of TMADH, revealed that the pKa of 8.4+/-0.1 for the wild-type enzyme-substrate complex represents ionization of the imidazolium side-chain of His-172. H172Q TMADH retains catalytic competence throughout the pH range investigated. At pH 10.5, and in contrast with the wild-type enzyme, flavin reduction in H172Q TMADH is biphasic. The fast phase is dependent on the trimethylamine concentration and exhibits a kinetic isotope effect of about 3; C-H bond cleavage is thus partially rate-limiting. In contrast, the slow phase does not show hyperbolic dependence on substrate concentration, and the observed rate shows no dependence on isotope, revealing that C-H bond cleavage is not rate-limiting. The analysis of H172Q TMADH, together with data recently acquired for the Y169F mutant of TMADH, reveals that C-H bond breakage is not initiated via abstraction of a proton from the substrate by an active-site base. The transfer of reducing equivalents to flavin via a carbanion mechanism is therefore unlikely.

Enzyme Activation↗
PubMed1999

The kinetic behavior of chicken liver sulfite oxidase.

A comprehensive kinetic study of sulfite oxidase has been undertaken over the pH range 6.0-10.0, including conventional steady-state work as well as rapid kinetic studies of both the reaction of oxidized enzyme with sulfite and reduced enzyme with cytochrome c (III). A comparison of the pH dependence of kcat, kred, and kox indicates that kred is principally rate limiting above pH 7, but that below this pH the pH dependence of kcat is influenced by that of kox. The pH independence of kred is consistent with our previous proposal concerning the reaction mechanism, in which attack of the substrate lone pair of electrons on a Mo(VI)O2 unit initiates the catalytic sequence. The pH dependence of kred/Kdsulfite indicates that a group on the enzyme having a pKa of approximately 9.3 must be deprotonated for effective reaction of oxidized enzyme with sulfite, possibly Tyr 322, which from the crystal structure of the enzyme constitutes part of the substrate binding site. There is no evidence for the HSO3-/SO32- pKa of approximately 7 in the pH profile for kred/Kdsulfite, suggesting that enzyme is able to oxidize the two equally well. By contrast, kcat/Kmsulfite and kred/Kdsulfite exhibit distinct pH dependence (the former is bell-shaped, the latter sigmoidal), again consistent with the oxidative half-reaction contributing to the kinetic barrier to catalysis at low pH. The pH dependence of kcat/Km(cyt c) (reflecting the second-order rate of reaction of free enzyme with free cytochrome) is bell-shaped and closely resembles that of kox/Kd(cyt c), reflecting the importance of the oxidative half-reaction in the low substrate concentration regime. The pH profile for kox/Kd(cyt c) indicates that two groups with a pKa of approximately 8 are involved in the reaction of free reduced enzyme with cytochrome c, one of which must be deprotonated and the other protonated. These results are consistent with the known electrostatic nature of the interaction of cytochrome c with its physiological partners.

Animals↗
PubMed1999

The reaction of trimethylamine dehydrogenase with trimethylamine.

The reductive half-reaction of trimethylamine dehydrogenase with its physiological substrate trimethylamine has been examined by stopped-flow spectroscopy over the pH range 6.0-11.0, with attention focusing on the fastest of the three kinetic phases of the reaction, the flavin reduction/substrate oxidation process. As in previous work with the slow substrate diethylmethylamine, the reaction is found to consist of three well resolved kinetic phases. The observed rate constant for the fast phase exhibits hyperbolic dependence on the substrate concentration with an extrapolated limiting rate constant (klim) greater than 1000 s-1 at pH above 8.5, 10 degrees C. The kinetic parameter klim/Kd for the fast phase exhibits a bell-shaped pH dependence, with two pKa values of 9.3 +/- 0.1 and 10. 0 +/- 0.1 attributed to a basic residue in the enzyme active site and the ionization of the free substrate, respectively. The sigmoidal pH profile for klim gives a single pKa value of 7.1 +/- 0. 2. The observed rate constants for both the intermediate and slow phases are found to decrease as the substrate concentration is increased. The steady-state kinetic behavior of trimethylamine dehydrogenase with trimethylamine has also been examined, and is found to be adequately described without invoking a second, inhibitory substrate-binding site. The present results demonstrate that: (a) substrate must be protonated in order to bind to the enzyme; (b) an ionization group on the enzyme is involved in substrate binding; (c) an active site general base is involved, but not strictly required, in the oxidation of substrate; (d) the fast phase of the reaction with native enzyme is considerably faster than observed with enzyme isolated from Methylophilus methylotrophus that has been grown up on dimethylamine; and (e) a discrete inhibitory substrate-binding site is not required to account for excess substrate inhibition, the kinetic behavior of trimethylamine dehydrogenase can be readily explained in the context of the known properties of the enzyme.

Catalysis↗
PubMed1999

The role of Tyr-169 of trimethylamine dehydrogenase in substrate oxidation and magnetic interaction between FMN cofactor and the 4Fe/4S center.

Tyr-169 in trimethylamine dehydrogenase is one component of a triad also comprising residues His-172 and Asp-267. Its role in catalysis and in mediating the magnetic interaction between FMN cofactor and the 4Fe/4S center have been investigated by stopped-flow and EPR spectroscopy of a Tyr-169 to Phe (Y169F) mutant of the enzyme. Tyr-169 is shown to play an important role in catalysis (mutation to phenylalanine reduces the limiting rate constant for bleaching of the active site flavin by about 100-fold) but does not serve as a general base in the course of catalysis. In addition, we are able to resolve two kinetically influential ionizations involved in both the reaction of free enzyme with free substrate (as reflected in klim/Kd), and in the breakdown of the Eox.S complex (as reflected in klim). In EPR studies of the Y169F mutant, it is found that the ability of the Y169F enzyme to form the spin-interacting state between flavin semiquinone and reduced 4Fe/4S center characteristic of wild-type enzyme is significantly compromised. The present results are consistent with Tyr-169 representing the ionizable group of pKa approximately 9.5, previously identified in pH-jump studies of electron transfer, whose deprotonation must occur for the spin-interacting state to be established.

Electron Spin Resonance Spectroscopy↗
PubMed1999

Nitric oxide-generated P420 nitric oxide synthase: characterization and roles for tetrahydrobiopterin and substrate in protecting against or reversing the P420 conversion.

The neuronal NO synthase (nNOS) heme binds self-generated NO, and this negatively regulates NO synthesis. Here we utilized the nNOS oxygenase domain and full-length nNOS along with various spectroscopic methods to (1) study formation of the six-coordinate ferrous NO complex and its conversion to a five-coordinate NO complex and (2) investigate the spectral and catalytic properties of the five-coordinate NO complex following its air oxidation to a ferric enzyme. NO bound quickly to ferrous nNOS oxygenase to form a six-coordinate NO complex (kon and koff values of 1.25 x 10(-)3 mM-1 s-1 and 128 s-1 at 10 degreesC, respectively) that was stable in the presence of L-arginine or tetrahydrobiopterin (BH4) but was converted to a five-coordinate NO complex in a biphasic process (k = 0.1 and 0.01 s-1 at 10 degreesC) in the absence of these molecules. Air oxidation of the ferrous six-coordinate NO complex generated an enzyme with full activity and ferrous-CO Soret absorbance at 444 nm. In contrast, oxidation of the five-coordinate NO complex generated an inactive dimer with ferrous-CO Soret absorbance at 420 nm, indicating nNOS was converted to a ferric P420 form. Incubation of ferric P420 nNOS with BH4 alone or BH4 and L-arginine resulted in time-dependent reactivation of catalysis and associated recovery of P450 character. Thus, nNOS is a heme-thiolate protein that can undergo a reversible P450-P420 conversion. BH4 has important roles in preventing P420 formation during NO synthesis, and in rescuing P420 nNOS.

Air↗
PubMed1999

The reductive half-reaction of xanthine oxidase. Reaction with aldehyde substrates and identification of the catalytically labile oxygen.

The kinetics of xanthine oxidase has been investigated with the aim of addressing several outstanding questions concerning the reaction mechanism of the enzyme. Steady-state and rapid kinetic studies with the substrate 2,5-dihydroxybenzaldehyde demonstrated that (kcat/Km)app and kred/Kd exhibit comparable bell-shaped pH dependence with pKa values of 6.4 +/- 0.2 and 8.4 +/- 0.2, with the lower pKa assigned to an active-site residue of xanthine oxidase (possibly Glu-1261, by analogy to Glu-869 in the crystallographically known aldehyde oxidase from Desulfovibrio gigas) and the higher pKa to substrate. Early steps in the catalytic sequence have been investigated by following the reaction of the oxidized enzyme with a second aldehyde substrate, 2-aminopteridine-6-aldehyde. The absence of a well defined acid limb in this pH profile and other data indicate that this complex represents an Eox.S rather than Ered.P complex (i.e. no chemistry requiring the active-site base has taken place in forming the long wavelength-absorbing complex seen with this substrate). It appears that xanthine oxidase (and by inference, the closely related aldehyde oxidases) hydroxylates both aromatic heterocycles and aldehydes by a mechanism involving base-assisted catalysis. Single-turnover experiments following incorporation of 17O into the molybdenum center of the enzyme demonstrated that a single oxygen atom is incorporated at a site that gives rise to strong hyperfine coupling to the unpaired electron spin of the metal in the MoV oxidation state. By analogy to the hyperfine interactions seen in a homologous series of molybdenum model compounds, we conclude that this strongly coupled, catalytically labile site represents a metal-coordinated hydroxide rather than the Mo=O group and that this Mo-OH represents the oxygen that is incorporated into product in the course of catalysis.

Aminopterin↗
PubMed1999

Strontium90 for determination of time since death.

Strontium90 (Sr90) is an artificial nuclear fission product of the atmospheric a-bomb testing between 1945 and 1979. It was spread throughout the atmosphere in the following years. Sr90 is an analogue to calcium and therefore enriched in human bones. Several studies especially in the 1960s and 1970s were undertaken to investigate the Sr90 burden and the resulting incorporated radiation in humans, but present studies are missing. In this study nine bone samples, three from 1931/32 and six from 1989 to 1994 were examined by measuring the Sr90 radiation. The samples from 1931/32 did not show any Sr90 activity. All the samples from 1989 and later showed a Sr90 activity, but the intensity was very variable. Subsequent investigations should be done to determine the cut-off year for measurable Sr90 activity. Furthermore the determination of a specific time since death depending on Sr90 activity should be possible, due to the ranging Sr90 pollution between 1950 and 1980 and different uptake in adolescents and adults.

Adolescent↗
PubMed1999

Molybdenum enzymes.

There are many molybdenum-containing enzymes distributed throughout the biosphere. The availability of molybdenum to biological systems is due to the high water solubility of oxidized forms of the metal. Molybdenum enzymes can be grouped on the basis of the structure of the metal centre. Three principal families of enzyme exist, with active sites consisting of (ppt)MoOS(OH) (the molybdenum hydroxylases), (ppt)MoO2(S-Cys) (the eukaryotic oxotransferases) and (ppt)2MoOX (the bacterial oxotransferases). Here, ppt represents a unique ppt cofactor (pyranopterin) that co-ordinates to the metal, and X is a metalliganded serine, cysteine or selenocysteine. The molybdenum hydroxylases catalyse their reactions differently to other hydroxylase enzymes, with water rather than molecular oxygen as the ultimate source of the oxygen atom incorporated into product, and with the generation rather than consumption of reducing equivalents. The active sites possess a catalytically labile Mo-OH (or possibly Mo-OH2) group that is transferred to substrate in the course of the hydroxylation reaction. These enzymes invariably have other redoxactive centres. The eukaryotic oxotransferases consist of the sulphite oxidases and plant nitrate reductases. They catalyse the transfer of an oxygen atom to or from their substrate (to and from nitrate) in a manner that involves formal oxidation-state changes of the molybdenum. As with the molybdenum hydroxylases, the ultimate source of oxygen is water rather than molecular oxygen. The bacterial oxotransferases and related enzymes differ from the other two groups of molybdenum enzymes in having two equivalents of the ppt cofactor co-ordinated to the metal. This family is quite diverse, as reflected in the fact that serine, cysteine or selenocysteine may be found co-ordinated to the molybdenum, depending on the enzyme. As in the case of the molybdenum hydroxylases, both eukaryotic and bacterial oxotransferases utilize water (rather than molecular oxygen) as the source of the oxygen atom incorporated into product, although for these enzymes, the catalytically labile oxygen in the active site is an Mo = O group rather than an Mo-OH.

Enzymes↗
PubMed1998

Formation of a tyrosyl radical in xanthine oxidase.

Treatment of xanthine oxidase with ferricenium at high pH gives rise to an EPR signal not previously seen with this enzyme. The signal is apparently isotropic at 9 GHz with a gavg of approximately 2 and once generated is stable to pH 6.0, so long as the sample is kept in the dark. Treatment of the signal-giving species with hydroxyurea results in complete loss of the signal, indicating that the signal is radical-based. Pretreatment of the enzyme with iodoacetate has no effect on signal formation with ferricenium. The ferricenium-generated EPR signal shows proton hyperfine coupling that is not lost upon exchange into D2O and bears considerable resemblance to the tyrosyl radical of the photosynthetic reaction center and other systems. These observations lead us to interpret the new ferricenium-generated EPR signal of xanthine oxidase as arising from a tyrosyl radical, the result of one-electron oxidation of a protein tyrosinate residue. Kinetic parameters for the reductive half-reaction of ferricenium-treated xanthine oxidase with xanthine were determined by stopped-flow spectrophotometry; kred and KDxanthine (15 s-1 and 12 microM, respectively) were essentially unchanged. Addition of 2-hydroxy-6-methylpurine (in the presence of 2 mg/mL catalase and superoxide dismutase) generated the "very rapid" MoV EPR signal while preserving the ferricenium-derived EPR signal, providing a further indication that the modified enzyme remains fully functional and the presence of the tyrosyl radical does not impact turnover by the enzyme. Coupling of the two signals was not evident, nor was coupling to the two 2Fe-2S centers or the flavin semiquinone evident. The implications of covalent modifications of proteins mediated by ferricenium are discussed.

Animals↗
PubMed1998

Adenosine 3':5'-cyclic monophosphate (cAMP)-inducible pyrimidine 5'-nucleotidase and pyrimidine nucleotide metabolism of chick embryonic erythrocytes.

Terminally differentiating erythrocytes degrade most of their RNA with subsequent release of mononucleotides. Pyrimidine mononucleotides are preferentially cleaved by an erythrocyte-specific pyrimidine 5'-nucleotidase; deficiency of this enzyme causes hemolytic anemia in humans. Details of the regulation of its activity during erythroid differentiation are unknown. The present study arose from the observation that the immature red blood cells (RBCs) of mid-term chick embryos contain high concentrations of uridine 5'-triphosphate (UTP) (5 to 6 mmol/L), which decline rapidly from days 13 to 14 onward. We analyzed two key enzymes of RBC pyrimidine nucleotide metabolism: pyrimidine nucleoside phosphorylase (PNP) and pyrimidine 5'-nucleotidase (P-5'-N), to evaluate if changes of enzyme activity during embryonic development are correlated with changes of RBC UTP. Secondly, we tested if these enzymes are under hormonal control. The results show that embryonic RBCs contain only minimal activity of PNP. In contrast, P-5'-N increases from day 13 on, suggesting that the enzyme is a limiting factor in UTP degradation. Activation of beta-adrenergic and A2A-adenosine receptors causes transcription-dependent de novo synthesis of P-5'-N. Because beta-adrenergic and adenosine receptors are also found on adult erythroid cells, P-5'-N might be an enzyme of differentiating RBCs whose expression is in part controlled by adenosine 3':5'-cyclic monophosphate (cAMP).

5'-Nucleotidase↗
PubMed1998

FTIR characterization of heterocycles lumazine and violapterin in solution: effects of solvent on anionic forms.

Fourier transform infrared (FTIR) spectra have been obtained from solution samples of the heterocycles uracil, lumazine, and violapterin and reveal interpretable carbonyl stretching frequencies. Spectra of conjugate bases of lumazine and violapterin demonstrate decreases in these carbonyl stretching frequencies upon ionization. Based on isotopic shifts from amide deuterated analogs, semiempirical QCFF/PI calculations were used to assign the vibrational frequencies in the region 1100-1800 cm-1 observed from samples in dimethylsulfoxide (DMSO) and aqueous solutions to specific normal modes. The observed deuterium shifts and the calculations suggest that, in some cases, N-H bending motions are coupled to the C=O stretching motions of the pyrimidine ring. These data suggest that for lumazine anions a change in solvent can significantly change the mixing of the N-H bending and C=O stretching vibrational motions. This implies that vibrational analysis for lumazine species in relatively noninteracting media like nonpolar solvents, mulls or pellets cannot necessarily be transferred to the system when it is dissolved in a polar, hydrogen-bonding solvent such as water. Although other explanations can be offered, our vibrational analysis suggests that the changes in normal mode composition of the predominantly C=O stretching vibrations of lumazine anion on going from dimethylsulfoxide to water solution are consistent with a change in the predominant tautomer of the heterocycle. This change appears to correspond to a shifting of the location of the remaining acidic proton to a different ring nitrogen atom. This interpretation is of interest in view of recent ab initio calculations which suggest that proton shifts may occur during the hydroxylation of lumazine as mediated by the enzyme xanthine oxidase.

Anions↗
PubMed1998

Population dose near the Semipalatinsk test site.

To determine the consequences of atmospheric atomic bomb tests for the population in the surroundings of the former nuclear weapons test site near Semipalatinsk in Kazakhstan, a pilot study was performed by an international cooperation between Kazakh, French, Czech and German institutions at two villages, Mostik and Maisk. Together with Kazakh scientists, eight experts from Europe carried out a field mission in September 1995 to assess, within the framework of a NATO supported project, the radiological situation as far as external doses, environmental contamination and body burden of man were concerned. A summary of the results obtained is presented. The actual radiological situation near the test site is characterized by fallout contaminations. Cs was found in upper soil layers in concentrations similar to those of the global fallout. Also Sr, Am and Co were observed. The resulting present dose to the population is low. Mean external doses from soil contamination for Maisk and Mostik (0.60-0.63 mSv/year) presently correspond to mean external doses in normal environments. Mean values of the annual internal doses observed in these two villages are below 2 microSv/year for 90Sr. For other radionuclides the internal doses are also negligible.

Biological Assay↗
PubMed1997

Reconstitution and characterization of a divergent plastocyanin from the photosynthetic prokaryote, Prochlorothrix hollandica, expressed in Escherichia coli.

Plastocyanin (PC) is a copper protein that serves as a mobile electron carrier between cytochrome f and Photosystem I in the light reactions of photosynthesis. Despite large variability in amino acid sequences and isoelectric points, PCs from cyanobacterial and chloroplast sources reveal considerable similarities with respect to their secondary and tertiary structures. In this paper, we have expressed in Escherichia coli a PC from the prokaryote Prochlorothrix hollandica, and efficiently reconstituted the protein with copper under conditions yielding the characteristics of a native holoPC, as judged by redox titration (Eo' = +376 mV), near and far UV circular dichroism, and electron paramagnetic resonance (EPR) spectroscopy. By comparison of amino acid sequences, P. hollandica PC is the most divergent homolog identified to date, and analysis of this reconstituted preparation may reveal new insights as to the structural requirements for electron transport between the PC copper center and neighboring reaction partners.

Circular Dichroism↗
PubMed1997

Involvement of a flavin iminoquinone methide in the formation of 6-hydroxyflavin mononucleotide in trimethylamine dehydrogenase: a rationale for the existence of 8alpha-methyl and C6-linked covalent flavoproteins.

In trimethylamine dehydrogenase, substrate is bound in the active site via cation-pi bonding to three aromatic residues (Tyr-60, Trp-264, and Trp-355). Mutation of one of these residues (Trp-355 --> Leu, mutant W355L) influences the chemistry of the flavin mononucleotide in the active site, enabling derivatization to 6-hydroxy-FMN. The W355L mutant is purified as a mixture of deflavo, natural 6-S-cysteinyl-FMN, and inactive 6-hydroxy-FMN forms, and the enzyme is severely compromised in its ability to oxidatively demethylate trimethylamine. Analysis of samples of the native and recombinant wild-type trimethylamine dehydrogenases also revealed the presence of 6-hydroxy-FMN, but at much reduced levels compared with that of the W355L enzyme. Unlike that for a C30A mutant of trimethylamine dehydrogenase, addition of substrate to the W355L trimethylamine dehydrogenase is not required for the production of 6-hydroxy-FMN. A mechanism is proposed for the 6-hydroxylation of FMN in trimethylamine dehydrogenase that involves an electrophilic flavin iminoquinone methide. The proposed mechanism involving the flavin iminoquinone methide could apply to the flavinylation of trimethylamine dehydrogenase at the C6 position but also to the flavinylation of enzymes via the 8alpha position, thus providing a rationale for the evolution of covalent flavoproteins in general. Covalent linkage at C6 or the 8alpha-methyl prevents 6-hydroxylation by direct modification at the C6 atom or by preventing formation of the flavin iminoquinone methide, respectively.

Binding Sites↗