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PubMed1976

Effects of pressure upon the fluorescence of the riboflavin binding protein and its flavin mononucleotide complex.

The effect of pressure in the range of 10(-3)-10 kbars upon the ultraviolet fluorescence of the riboflavin binding protein and the fluorescences of its complex with flavin mononucleotide has been studied. The fluorescence spectrum of the isolated protein showed a reversible red shift of 12nm (1000 cm-1) at high pressure, indicating the reversible exposure of the tryptophan to solvent. From the pressure dependence of the visible fluorescence of the protein-flavin complex in the region of 1-4 kbars the volume change in dissociation of the protein-ligand complex was estimated to be +3.3ml/mol. A very sharp increase in fluorescence-up to 30-fold of the low-pressure value-takes place in the region 5-8 kbars. This increase is due to release of the flavin from the complex and is assigned to pressure denaturation of the protein. The midpoint, rho 1/2, of this transition was found at 6.5 kbars and the change in volume, delta, in the reaction (native-to-denatured) was calculated to be -74ml/mol. Addition of up to 30% methanol results in a progressive decrease both in delta and rho 1/2, in agreement with the concept that hydrophobic bonding stabilizes the native structure.

Bromosuccinimide↗
PubMed1975

Photoexcited bacterial luminescence. Spectral properties and mechanistic implication of a reduced flavine-like prosthetic group associated with photoexcitable luciferase.

A prosthetic group, designated B, has been isolated from bacterial photoexcitable luciferase and found to possess spectral and photochemical properties characteristic of substituted reduced flavines. Its fluorescence when bound to luciferase has an excitation maximum at 375 nm, correlating well with the absorption spectrum, and an emission peaking at 495 nm. However, free B is nonfluorescent in aqueous solution at ambient temperature. Both free and luciferase-bound B show similar negative circular dichroism in the region 330-475 nm with troughs at 375 and 380 nm, respectively. In the luciferase reaction initiated by FMNH2, B is an inhibitor competitive with FMNH2. Irradiation of photoexcitable luciferase converts B to FMN, the latter identified spectrally, enzymatically, and chromatographically. These findings lead to the suggestion that B is a substituted FMNH2. The luciferase-bound B resembles but is not identical with the normal flavine intermediate obtainable by reacting luciferase with reduced flavine mononucleotide and oxygen. It is hypothesized that B is a false intermediate of the bacterial bioluminescence reaction, and a mechanism for the photoexcited bioluminescence reaction is suggested.

Binding Sites↗
PubMed2001

Role of hydrogen bonding interactions to N(3)H of the flavin mononucleotide cofactor in the modulation of the redox potentials of the Clostridium beijerinckii flavodoxin.

The role of the hydrogen bonding interaction with the N(3)H of the flavin cofactor in the modulation of the redox properties of flavoproteins has not been extensively investigated. In the flavodoxin from Clostridium beijerinckii, the gamma-carboxylate group of glutamate-59 serves as a dual hydrogen bond acceptor with the N(3)H of flavin mononucleotide (FMN) cofactor and the amide hydrogen of the adjacent polypeptide backbone in all three oxidation states. This "bridging" interaction serves to anchor the FMN in the binding site, which, based on the E59Q mutant, indirectly affects the stability of the neutral flavin semiquinone by facilitating a strong and critical interaction at the FMN N(5)H [Bradley, L. H., and Swenson, R. P. (1999) Biochemistry 38, 12377-12386]. In this study, the specific role of the N(3)H interaction itself was investigated through the systematic replacement of Glu59 by aspartate, asparagine, and alanine in an effort to weaken, disrupt, and/or eliminate this interaction, respectively. Just as for the E59Q mutant, each replacement significantly weakened the binding of the cofactor, particularly for the semiquinone state, affecting the midpoint potentials of each one-electron couple in opposite directions. (1)H-(15)N HSQC nuclear magnetic resonance (NMR) spectroscopic studies revealed that not only was the N(3)H interaction weakened as anticipated, but so also was the hydrogen bonding interaction with the N(5)H. Using the temperature coefficients of the N(5)H to quantify and correct for changes in this interaction, the contribution of the N(3)H hydrogen bond to the binding of each redox state of the FMN was isolated and estimated. Based on this analysis, the N(3)H hydrogen bonding interaction appears to contribute primarily to the stability of the oxidized state (by as much as 2 kcal/mol) and to a lesser extent the reduced states. It is concluded that this interaction contributes only modestly (<45 mV) to the modulation of the midpoint potential for each redox couple in the flavodoxin. These conclusions are generally consistent with ab initio calculations and model studies on the non-protein-bound cofactor.

Amino Acid Substitution↗
PubMed2002

Flavin thermodynamics explain the oxygen insensitivity of enteric nitroreductases.

Bacterial nitroreductases are NAD(P)H-dependent flavoenzymes which catalyze the oxygen-insensitive reduction of nitroaromatics, quinones, and riboflavin derivatives. Despite their broad substrate specificity, their reactivity is very specific for two-electron, not one-electron, chemistry. We now describe the thermodynamic properties of the flavin mononucleotide cofactor of Enterobacter cloacae nitroreductase (NR), determined under a variety of solution conditions. The two-electron redox midpoint potential of NR is -190 mV at pH 7.0, and both the pH dependence of the midpoint potential and the optical spectrum of the reduced enzyme indicate that the transition is from neutral oxidized flavin to anionic flavin hydroquinone. The one-electron-reduced semiquinone states of both the free enzyme and an NR-substrate analogue complex are strongly suppressed based on optical spectroscopy and electron paramagnetic resonance measurements. This can explain the oxygen insensitivity of NR and its homologues, as it makes the execution of one-electron chemistry thermodynamically unfavorable. Therefore, we have established a chemical basis for the recent finding that a nitroreductase is a member of the soxRS oxidative defense regulon in Escherichia coli [Liochev, S. I., Hausladen, A., Fridovich, I. (1999) Proc. Natl. Acad. Sci. U.S.A. 96 (7), 3537-3539]. We also report binding affinities for the FMN cofactor in all three oxidation states either determined fluorometrically or calculated using thermodynamic cycles. Thus, we provide a detailed picture of the thermodynamics underlying the unusual activity of NR.

Apoenzymes↗
PubMed2003

Characterization of a flavin radical product in a C57M mutant of a LOV1 domain by electron paramagnetic resonance.

In the flavin mononucleotide-binding LOV1 domain of the Phot1-receptor from Chlamydomonas reinhardtii the photoreactive cysteine C57 has been replaced by methionine. Photoexcitation of this C57M mutant yields a metastable photoproduct (C57M-415) that thermally decomposes into a stable paramagnetic species (C57M-675) with extremely red-shifted absorption in the visible range. In this contribution, we describe the characterization of this radical by multi-frequency electron paramagnetic resonance and electron-nuclear double resonance. The main features of the spectra identify the paramagnetic species as a flavin neutral radical. However, detailed analysis shows that the isoalloxazine moiety of the flavin is alkyl substituted at N(5), rather than protonated as is usually the case. The implication of these observations on the likely mechanism of photoproduct generation in wild-type LOV domains is discussed.

Amino Acid Substitution↗
PubMed2003

Irreversible photoreduction of flavin in a mutated Phot-LOV1 domain.

Phot photoreceptors make up an important protein family regulating biological processes in response to blue light. They contain two light, oxygen, and voltage sensitive (LOV) domains and a serine/threonine kinase domain. Both LOV domains noncovalently bind a flavin mononucleotide (FMN). Upon absorption of blue light, the LOV domains undergo a photocycle, transiently forming a covalent adduct of a cysteine residue and the FMN (LOV-390). The mechanism of formation of this flavin-thiol adduct is still unclear. We studied a mutant of the LOV1 domain from the green alga Chlamydomonas reinhardtii with a methionine replacing the reactive cysteine 57 (C57M). As in the wild type, irradiation leads to formation of a photoadduct, which, however, is irreversibly converted into a red absorbing species, C57M-675. On the basis of spectroscopic results and the 2.1 A resolution crystal structure, this highly unusual FMN species was assigned to a neutral flavin radical covalently attached to the apoprotein at the N(5) position. In contrast to other flavoprotein neutral radicals, C57M-675 is stable even under aerobic or denaturing conditions. Pathways for the photoinduced formation of the adduct are discussed for the C57M mutant as well as the wild-type LOV1 domain.

Animals↗
PubMed2005

The role of Val-265 for flavin adenine dinucleotide (FAD) binding in pyruvate oxidase: FTIR, kinetic, and crystallographic studies on the enzyme variant V265A.

In pyruvate oxidase (POX) from Lactobacillus plantarum, valine 265 participates in binding the cofactor FAD and is responsible for the strained conformation of its isoalloxazine moiety that is visible in the crystal structure of POX. The contrasting effects of the conservative amino acid exchange V265A on the enzyme's catalytic properties, cofactor affinity, and protein structure were investigated. The most prominent effect of the exchange was observed in the 2.2 A crystal structure of the mutant POX. While the overall structures of the wild-type and the variant are similar, flavin binding in particular is clearly different. Local disorder at the isoalloxazine binding site prevents modeling of the complete FAD cofactor and two protein loops of the binding site. Only the ADP moiety shows well-defined electron density, indicating an "anchor" function for this part of the molecule. This notion is corroborated by competition experiments where ADP was used to displace FAD from the variant enzyme. Despite the fact that the affinity of FAD binding in the variant is reduced, the catalytic properties are very similar to the wild-type, and the redox potential of the bound flavin is the same for both proteins. The rate of electron transfer toward the flavin during turnover is reduced to one-third compared to the wild-type, but k(cat) remains unchanged. Redox-triggered FTIR difference spectroscopy of free FAD shows the nu(C(10a)=N(1)) band at 1548 cm(-)(1). In POX-V265A, this band is found at 1538 cm(-)(1) and thus shifted less strongly than in wild-type POX where it is found at 1534 cm(-)(1). Taking these observations together, the conservative exchange V265A in POX has a surprisingly small effect on the catalytic properties of the enzyme, whereas the effect on the three-dimensional structure is rather big.

Base Sequence↗
PubMed2004

Aminobacter aminovorans NADH:flavin oxidoreductase His140: a highly conserved residue critical for NADH binding and utilization.

Homodimeric FRD(Aa) Class I is an NADH:flavin oxidoreductase from Aminobacter aminovorans. It is unusual because it contains an FMN cofactor but utilizes a sequential-ordered kinetic mechanism. Because little is known about NADH-specific flavin reductases in general and FRD(Aa) in particular, this study aimed to further explore FRD(Aa) by identifying the functionalities of a key residue. A sequence alignment of FRD(Aa) with several known and hypothetical flavoproteins in the same subfamily reveals within the flavin reductase active-site domain a conserved GDH motif, which is believed to be responsible for the enzyme and NADH interaction. Mutation of the His140 in this GDH motif to alanine reduced FRD(Aa) activity to <3%. An ultrafiltration assay and fluorescence quenching demonstrated that H140A FRD(Aa) binds FMN in the same 1:1 stoichiometric ratio as the wild-type enzyme, but with slightly weakened affinity (K(d) = 0.9 microM). Anaerobic stopped-flow studies were carried out using both the native and mutated FRD(Aa). Similar to the native enzyme, H140A FRD(Aa) was also able to reduce the FMN cofactor by NADH although much less efficiently. Kinetic analysis of anaerobic reduction measurements indicated that the His140 residue of FRD(Aa) was essential to NADH binding, as well as important for the reduction of the FMN cofactor. For the native enzyme, the cofactor reduction was followed by at least one slower step in the catalytic pathway.

Alphaproteobacteria↗
PubMed1996

Binding of the oxidized, reduced, and radical flavin species to chorismate synthase. An investigation by spectrophotometry, fluorimetry, and electron paramagnetic resonance and electron nuclear double resonance spectroscopy.

Chorismate synthase (EC 4.6.1.4) binds oxidized riboflavin-5'-phosphate mononucleotide (FMN) with a KD of 30 microM at 25 degrees C, but in the presence of 5-enolpyruvylshikimate-3-phosphate (EPSP), the KD decreases to ca. 20 nM. Similar effects occur with the substrate analogue (6R)-6-fluoro-EPSP (KD = 36 nM) and chorismate (KD = 540 nM). Fluorescence of oxidized FMN is slightly quenched in the presence of chorismate synthase. Addition of EPSP or the (6R)-6-fluoro analogue causes a shift of the fluorescence from 520 to 495 nm. Chorismate causes no shift in, but a quenching of, the fluorescence emission maximum. In the presence of EPSP, (6R)-6-fluoro-EPSP, or chorismate, the neutral flavinsemiquinone is generated. The electron paramagnetic resonance (EPR) line width of the flavin radical is indicative of a neutral flavinsemiquinone. Frozen solution electron nuclear double resonance (ENDOR) of the radical with (6R)-6-fluoro-EPSP shows a number of proton ENDOR line pairs. The largest splitting is assigned to a hyperfine coupling to the methyl group beta-protons at position 8 of the isoalloxazine ring. The hyperfine-coupling (hfc) components have values of A perpendicular = 8.07 MHz and A parallel = 9.60 MHz, giving Aiso of 8.58 MHz, consistent with a neutral semiquinone form. The isotropic hfc coupling of the 8-methyl protons with (6R)-6-fluoro-EPSP decreases by about 0.5 MHz when chorismate is bound, indicating that the spin density distribution within the isoalloxazine ring system depends critically on the nature of the ligand. The redox potential of FMN in the presence of chorismate synthase was 95 mV more positive than that of free FMN (at pH 7.0), equivalent to a 1660-fold tighter binding of reduced FMN. The pH dependence of the redox potential of chorismate synthase-bound FMN exhibits a slope of -30 mV per pH unit between pH 6 and 9, indicating that the two-electron reduction of the flavin is associated with the uptake of one proton; this, and the UV-visible spectrum, is consistent with the reduced flavin being bound to chorismate synthase in its monoanionic form.

Electron Spin Resonance Spectroscopy↗
PubMed1997

Structure of bacterial luciferase beta 2 homodimer: implications for flavin binding.

The crystal structure of the beta 2 homodimer of Vibrio harveyi luciferase has been determined to 2.5 A resolution by molecular replacement. Crystals were grown serendipitously using the alpha beta form of the enzyme. The subunits of the homodimer share considerable structural homology to the beta subunit of the alpha beta luciferase heterodimer. The four C-terminal residues that are disordered in the alpha beta structure are fully resolved in our structure. Four peptide bonds have been flipped relative to their orientations in the beta subunit of the alpha beta structure. The dimer interface of the homodimer is smaller than the interface of the heterodimer in terms of buried surface area and number of hydrogen bonds and salt links. Inspection of the subunits of our structure suggests that FMNH2 cannot bind to the beta 2 enzyme at the site that has been proposed for the alpha beta enzyme. However, we do uncover a potential FMNH2 binding pocket in the dimer interface, and we model FMN into this site. This proposed flavin binding motif is consistent with several lines of biochemical and structural evidence and leads to several conclusions. First, only one FMNH2 binds per homodimer. Second, we predict that reduced FAD and riboflavin should be poor substrates for beta 2. Third, the reduced activity of beta 2 compared to alpha beta is due to solvent exposure of the isoalloxazine ring in the beta 2 active site. Finally, we raise the question of whether our proposed flavin binding site could also be the binding site for flavin in the alpha beta enzyme.

Binding Sites↗
PubMed1999

The midpoint potentials for the oxidized-semiquinone couple for Gly57 mutants of the Clostridium beijerinckii flavodoxin correlate with changes in the hydrogen-bonding interaction with the proton on N(5) of the reduced flavin mononucleotide cofactor as measured by NMR chemical shift temperature dependencies.

In the Clostridium beijerinckii flavodoxin, the reduction of the flavin mononucleotide (FMN) cofactor is accompanied by a local conformation change in which the Gly57-Asp58 peptide bond "flips" from primarily the unusual cis O-down conformation in the oxidized state to the trans O-up conformation such that a new hydrogen bond can be formed between the carbonyl group of Gly57 and the proton on N(5) of the neutral FMN semiquinone radical [Ludwig, M. L., Pattridge, K. A., Metzger, A. L., Dixon, M. M., Eren, M., Feng, Y., and Swenson, R. P. (1997) Biochemistry 36, 1259-1280]. This interaction is thought to contribute to the relative stabilization of the flavin semiquinone and may be at least partially responsible for the substantial separation of the midpoint potentials of the two one-electron reduction steps. Through a series of amino acid substitutions, the above cited study demonstrated the critical role of the often conserved glycine residue in this process. However, it has not been directly established experimentally as to whether these substitutions brought about the changes in the midpoint potentials by altering the strength of this hydrogen-bonding interaction as proposed. In this study, the relative strengths of the FMN N(5)H.O57 hydrogen bond in wild type and the G57A, G57N, and G57T mutants were evaluated by measuring the temperature dependency of the chemical shift for the proton on N(5) of the fully reduced cofactor by 1H-15N HSQC nuclear magnetic resonance spectroscopy. Based on the established correlation between the temperature coefficient of amide protons and the strength of hydrogen bonding in small peptides, the apparent strength of the N(5)H.O57 interaction was found to depend on the properties of the side chain at position 57. The glycine residue found in the wild-type flavodoxin appears to provide the strongest interaction while the beta-branched side chain in the G57T mutant provides the weakest. A good correlation was noted between the temperature coefficients of N(5)H and the one-electron reduction potential for the ox/sq couple as well as the binding free energy of the FMN semiquinone in this group of mutants. These results provide more direct quantitative evidence that support the previous hypothesis that this conformation change and the associated formation of the hydrogen bonding interaction with N(5)H of the reduced FMN represent an important means of stabilizing the neutral semiquinone and in modulating the oxidation-reduction potentials of the flavin cofactor in this and perhaps other flavodoxins.

Amino Acid Substitution↗
PubMed1999

Control of luminescence decay and flavin binding by the LuxA carboxyl-terminal regions in chimeric bacterial luciferases.

Bacterial luciferases (LuxAB) can be readily classed as slow or fast decay luciferases based on their rates of luminescence decay in a single turnover assay. Luciferases from Vibrio harveyi and Xenorhabdus (Photorhabdus) luminescens have slow decay rates, and those from the Photobacterium genus, such as P. (Vibrio) fischeri, P. phosphoreum, and P. leiognathi, have rapid decay rates. By generation of an X. luminescens-based chimeric luciferase with a 67 amino acid substitution from P. phosphoreum LuxA in the central region of the LuxA subunit, the "slow" X. luminescens luciferase was converted into a chimeric luciferase, LuxA(1)B, with a significantly more rapid decay rate. Two other chimeras with P. phosphoreum sequences substituted closer to the carboxyl terminal of LuxA, LuxA(2)B and LuxA(3)B, retained the characteristic slow decay rates of X. luminescens luciferase but had weaker interactions with both reduced and oxidized flavins, implicating the carboxyl-terminal regions in flavin binding. The dependence of the luminescence decay on concentration and type of fatty aldehyde indicated that the decay rate of "fast" luciferases arose due to a high dissociation constant (K(a)) for aldehyde (A) coupled with the rapid decay of the resultant aldehyde-free complex via a dark pathway. The decay rate of luminescence (k(T)) was related to the decanal concentration by the equation: k(T) = (k(L)A + k(D)K(a))/(K(a) + A), showing that the rate constant for luminescence decay is equal to the decay rate via the dark- (k(D)) and light-emitting (k(L)) pathways at low and high aldehyde concentrations, respectively. These results strongly implicate the central region in LuxA(1)B as critical in differentiating between "slow" and "fast" luciferases and show that this distinction is primarily due to differences in aldehyde affinity and in the decomposition of the luciferase-flavin-oxygen intermediate.

Aldehydes↗
PubMed2002

NADH model systems functionalized with Zn(II)-cyclen as flavin binding site-structure dependence of the redox reaction within reversible aggregates.

The relative positions and conformations of the prosthetic group FAD and the cofactor NADH have been remarkably conserved within the structurally diverse group of flavin enzymes. To provide a chemical rational for such an obviously optimal relative disposition of the redox partners for efficient reaction we have synthesized NADH models with Zn(II)-cyclen substituents for reversible flavin binding in water. Altogether, four of these model systems with systematically varying spacer length between the recognition site and the redox active dihydronicotinamide were prepared. The binding of these model systems to riboflavin tetraacetate was confirmed by potentiometric pH titration in water and their reaction with flavin was followed by UV-vis spectroscopy in aqueous media under physiological conditions. The measurements reveal a significant rate enhancement of up to 175 times that of an intermolecular reaction. Moreover, a strong dependence of the reaction rate on the spacer length was observed, which clearly shows that within the dynamic reversible assembly only the optimal relative disposition of the redox partners ensures an efficient redox reaction.

Binding Sites↗
PubMed2005

Femtosecond dynamics of flavin cofactor in DNA photolyase: radical reduction, local solvation, and charge recombination.

We report here our femtosecond studies of the photoreduction dynamics of the neutral radical flavin (FADH) cofactor in E. coli photolyase, a process converting the inactive form to the biologically active one, a fully reduced deprotonated flavin FADH(-). The observed temporal absorption evolution revealed two initial electron-transfer reactions, occurring in 11 and 42 ps with the neighboring aromatic residues of W382 and F366, respectively. The new transient absorption, observed at 550 nm previously in photolyase, was found from the excited-state neutral radical and is probably caused by strong interactions with the adenine moiety through the flavin U-shaped configuration and the highly polar/charged surrounding residues. The solvation dynamics from the locally ordered water molecules in the active site was observed to occur in approximately 2 ps. These ultrafast ordered-water motions are critical to stabilizing the photoreduction product FADH(-) instantaneously to prevent fast charge recombination. The back electron-transfer reaction was found to occur in approximately 3 ns. This slow process, consistent with ultrafast stabilization of the catalytic cofactor, favors photoreduction in photolyase.

Binding Sites↗
PubMedDate not supplied

Coenzyme models. 10. Rapid oxidation of NADH by a flavin immobilized in cationic polyelectrolytes.

The title flavin rapidly oxidizes NADH, the observed enhancement being of 460--4650-fold compared with 3-methyltetra-O-acetylriboflavin. Analysis of the kinetic data at 30 degrees C established that the reaction proceeds according to the Michaelis-Menten kinetics at low ionic strength (mu = 0.02: Km = ca. 10(-5) M, kact. = 0.06-0.37 s-1) and according to the simple second-order kinetics at high ionic strength (mu = 0.3), the largest rate difference being 342-fold. On the other hand, the oxidation of 1-benzyl-1,4-dihydronicotinamide, a NADH model compound, was not accelerated by the use of the title flavin. It is concluded, therefore, that the formation of the polymeric flavin-NADH complexes due to the electrostatic interaction is responsible for the marked rate enhancement. This is the first example that the oxidation of NADH is facilitated in the nonenzymatic system.

Flavins↗
PubMed1985

Identification of the covalently bound flavins of D-gluconate dehydrogenases from Pseudomonas aeruginosa and Pseudomonas fluorescens and of 2-keto-D-gluconate dehydrogenase from Gluconobacter melanogenus.

An improved method is presented for the purification of 8 alpha-(N1-histidyl)riboflavin, 8 alpha-(N3-histidyl)riboflavin and their 2',5'-anhydro forms, which permits the isolation of sizeable quantities of each of these compounds from a synthetic mixture in pure form. Flavin peptides were isolated from the D-gluconate dehydrogenases of Pseudomonas aeruginosa and Pseudomonas fluorescens and from the 2-keto-D-gluconate dehydrogenase of Gluconobacter melanogenus. After conversion into the aminoacyl-riboflavin, the flavin in all three enzymes was identified as 8 alpha-(N3-histidyl)riboflavin. By sequential treatment with nucleotide pyrophosphatase and alkaline phosphatase, the flavin in each enzyme was shown to be in the dinucleotide form.

Carbohydrate Dehydrogenases↗
PubMed1993

Expression in Escherichia coli of the flavin and the haem domains of Hansenula anomala flavocytochrome b2 (flavodehydrogenase and b2 core) and characterization of the recombinant proteins.

The flavin and haem domains of Hansenula anomala flavocytochrome b2 have been independently expressed in Escherichia coli. The flavin domain activity, studied only in the total cellular extract, owing to its instability, has characteristics very similar to those of the flavin domain obtained by proteolysis. The haem domain (r-core) has been purified to homogeneity and characterized in detail from spectroscopic and functional points of view. Spectral differences with respect to the domain produced by proteolysis (p-core) were found using resonance Raman and c.d. spectroscopy and have been interpreted in terms of changes in haem-protein interactions. However, this structural difference is functionally silent, since the r-core is able to reduce cytochrome c with the same efficiency as the proteolytic domain.

Base Sequence↗
PubMed1998

L-Mandelate dehydrogenase from Rhodotorula graminis: cloning, sequencing and kinetic characterization of the recombinant enzyme and its independently expressed flavin domain.

The l-mandelate dehydrogenase (L-MDH) from the yeast Rhodotorula graminis is a mitochondrial flavocytochrome b2 which catalyses the oxidation of mandelate to phenylglyoxylate coupled with the reduction of cytochrome c. We have used the N-terminal sequence of the enzyme to isolate the gene encoding this enzyme using the PCR. Comparison of the genomic sequence with the sequence of cDNA prepared by reverse transcription PCR revealed the presence of 11 introns in the coding region. The predicted amino acid sequence indicates a close relationship with the flavocytochromes b2 from Saccharomyces cerevisiae and Hansenula anomala, with about 40% identity to each. The sequence shows that a key residue for substrate specificity in S. cerevisiae flavocytochrome b2, Leu-230, is replaced by Gly in L-MDH. This substitution is likely to play an important part in determining the different substrate specificities of the two enzymes. We have developed an expression system and purification protocol for recombinant L-MDH. In addition, we have expressed and purified the flavin-containing domain of L-MDH independently of its cytochrome domain. Detailed steady-state and pre-steady-state kinetic investigations of both L-MDH and its independently expressed flavin domain have been carried out. These indicate that L-MDH is efficient with both physiological (cytochrome c, kcat=225 s-1 at 25 degrees C) and artificial (ferricyanide, kcat=550 s-1 at 25 degrees C) electron acceptors. Kinetic isotope effects with [2-2H]mandelate indicate that H-C-2 bond cleavage contributes somewhat to rate-limitation. However, the value of the isotope effect erodes significantly as the catalytic cycle proceeds. Reduction potentials at 25 degrees C were measured as -120 mV for the 2-electron reduction of the flavin and -10 mV for the 1-electron reduction of the haem. The general trends seen in the kinetic studies show marked similarities to those observed previously with the flavocytochrome b2 (L-lactate dehydrogenase) from S. cerevisiae.

Alcohol Oxidoreductases↗