Flavocytochrome enzyme

This enzyme shares structural and mechanistic properties with VAOinl In contrast to VAO it is not an oxidase as regeneration of the covalently bound FAD with molecular oxygen is not possible. It is a flavocytochrome enzyme. The reduction equivalents from the substrate are transferred to a type c cytochrome156, 57]. In... 

L-lactate-cytochrome c-oxidoreductase (flavocytochrome was isolated for the first time from the thermo-tolerant yeast H. polymorpha. The mentioned above enzyme preparations were used for construction of the biorecognition elements of electrochemical sensors. 

The oxidation by strains of Pseudomonas putida of the methyl group in arenes containing a hydroxyl group in the para position is, however, carried out by a different mechanism. The initial step is dehydrogenation to a quinone methide followed by hydration (hydroxylation) to the benzyl alcohol (Hopper 1976) (Figure 3.7). The reaction with 4-ethylphenol is partially stereospecific (Mclntire et al. 1984), and the enzymes that catalyze the first two steps are flavocytochromes (Mclntire et al. 1985). The role of formal hydroxylation in the degradation of azaarenes is discussed in the section on oxidoreductases (hydroxylases). 

A first application using ferroceneboronic acid as mediator [45] was described for the transformation of p-hydroxy toluene to p-hydroxy benzaldehyde which is catalyzed by the enzyme p-cresolmethyl hydroxylase (PCMH) from Pseudomonas putida. This enzyme is a flavocytochrome containing two FAD and two cytochrome c prosthetic groups. To develop a continuous process using ultrafiltration membranes to retain the enzyme and the mediator, water soluble polymer-bound ferrocenes [50] such as compounds 3-7 have been applied as redox catalysts for the application in batch electrolyses (Fig. 12) or in combination with an electrochemical enzyme membrane reactor (Fig. 13) [46, 50] with excellent results. 

The success of Chapman and co-workers in expression of flavocytochrome 2 in E. coli [23] is encouraging in its impUcations for future expression of flavoproteins in this host because, in their experience both the flavin and heme groups are incorporated into the recombinant protein. Moreover, the bacterial expression system produces the protein 500-1000 fold more efficiently than the yeast from which it was cloned. The enzyme produced in E. coli, however, lacks the first five amino acid residues at its amino terminus, a result which presumably reflects subtle differences in protein synthesis between the two organisms. 

In contrast to the flavin oxidases, flavin dehydrogenases pass electrons to carriers within electron transport chains and the flavin does not react with 02. Examples include a bacterial trimethylamine dehydrogenase (Fig. 15-9) which contains an iron-sulfur duster that serves as the immediate electron acceptor167 169 and yeast flavocytochrome b2, a lactate dehydrogenase that passes electrons to a built-in heme group which can then pass the electrons to an external acceptor, another heme in cytochrome c.170-173 Like glycolate oxidase, these enzymes bind their flavin coenzyme at the ends of 8-stranded a(i barrels similar... 

Flavocytochrome b2 catalyses the oxidation of lactate to pyruvate at the expense of cytochrome C. After reduction of flavin (FMN) by the substrate, reducing equivalents are transferred to heme b2 and from there to cytochrome C602. The mechanism of this process has been studied603 at 5.0 °C by determining the D KIE in the FMN reduction using L-[2-2H]lactate and wild-type enzyme and also with the Y143F mutant prepared from transformed Escherichia coli604. Tritium IE in the conversion of [2-3-H]lactate to... 

Flavocytochrome b2 from Saccharomyces cerevisiae, a member of the FMN-dependent oxidoreductase superfamily, catalyzes the two-electron oxidation of lactate to pyruvate with subsequent electron-transfer to cytochrome c via the bound flavin [55], What distinguishes the enzyme from other family members is the N-terminal fusion of a heme-binding domain to the ySa-barrel structure, which hosts the primary active site. Rather than dumping the electrons from the reduced flavin hydroquinone onto molecular oxygen, they are transferred intramolecularly to the heme-binding domain and from there in a second intermolecular step to cytochrome c. 

Cunane, L. M., Chen, Z.-w., Shamala, N., Mathews, F. S., Cronin, C. N., and Mclntire, W. S., 2000, Structures of the flavocytochrome p-cresol methylhydroxylase and its enzyme-substrate complex gated substrate entry and proton relays support the proposed catalytic mechanism. J. Mol. Biol. 295 357n374. 

Hazzard, J. T., McDonough, C. A., and Tollin, G., 1994, Intramolecular electron transfer in yeast flavocytochrome b2 upon one-electron photooxidation of the fully reduced enzyme evidence for redox state control of heme-flavin communication. Biochemistry 33 13445nl3454. 

Koerber, S. C., Mclntire, W. S., Bohmont, C., and Singer, T. P., 1985, Resolution of the flavocytochrome p-cresol methylhydroxylase into subunits and reconstitution of the enzyme. Biochemistry 24 5276n5280. 

Flavocytochromes 2 2-hydroxyacid dehydrogenases found in the inter-membrane space of yeast mitochondria where they couple oxidation of the substrate to reduction of cytochrome c. Examples include the enzymes from Saccharomyces cerevisiae and Hansenula anomala, both of which are l-lactate dehydrogenases (Chapman et al., 1998), and the enzyme from Rhodotorula graminis which is a L-mandelate dehydrogenase (Ilias et al., 1998). This article will concentrate on the flavocytochrome 2 (L-lactate cytochrome c oxidoreductase) from S. cerevisiae (Bakersi yeast), since this is by far the most studied of these enzymes (Chapman et al., 1991). Therefore, throughout this article, the term flavocytochrome 2 will refer specifically to the enzyme from S. cerevisiae unless otherwise stated. 

As a respiratory enzyme, the production of flavocytochrome bi is induced by the presence of oxygen and, more specifically, L-lactate. In addition to providing pyruvate (the product of lactate oxidation) for the Krebs cycle, flavocytochrome bj also participates in a shorter respiratory chain that ultimately directs the energy gained from L-lactate dehydrogenation... 

Electron flow through flavocytochrome bz has been extensively studied in both the S. cerevisiae (Tegoni et al., 1998 Daff et al., 1996a Chapman et al., 1994 Pompon, 1980) and H. anomala (CapeillEre-Blandin et al., 1975) enzymes. The catalytic cycle is shown in Figure 3. Firstly, the flavin is reduced by L-lactate a carbanion mechanism has been proposed for this redox step (Lederer, 1991). Complete (two-electron) reduction of the flavin is followed by intra-molecular electron transfer from fully-reduced flavin to heme, generating flavin semiquinone and reduced heme (Daff et al.. 

The fundamentals on which the carbanion mechanism is founded are the early studies on the related enzj mes D-amino acid oxidase (Walsh et al., 1972 and 1971), lactate oxidase (Walsh et al., 1973) and flavocytochrome bj (Urban and Lederer, 1985 Pompon and Lederer, 1985). It is interesting that all of the key work, which established the carbanion mechanism, was done before any 3-dimensional structures were available on the respective enzymes. 

The core requirement for the carbanion mechanism to operate is that an active-site base must abstract the a-carbon hydrogen of the substrate, as a proton, forming a carbanion intermediate (Lederer, 1991). This would then require the equivalent of two electrons to be transferred to the flavin either with or without the formation of a covalent intermediate between the a-carbon and the flavin N-5 (Ghisla and Massey, 1989). With this in mind, it is intriguing to find that the crystal structure of D-amino acid oxidase reveals that there is no residue correctly located to act as the active-site base required for the carbanion mechanism (Mattevi et al., 1996 Mizu-tani et al., 1996). In fact, the crystallographic information available is far more consistent with this enzyme operating a hydride transfer mechanism (Mattevi et al., 1996). If this is correct then the earlier experiments on d-amino acid oxidase, which were claimed to be diagnostic of a carbanion mechanism, are ealled into question. It is important to note that similar experiments were used to provide support for a carbanion mechanism in the ease of flavocytochrome b2-... 

The high-level expression of recombinant flavocytochrome 7>2 in E. call (Black et al., 1989) has allowed the active site of the enzyme to be probed using site-directed mutagenesis. Two particular residues, His373 and Tyr254, have been examined in detail, since they have important roles in catalysis. The substitution of His373 by glutamine resulted in an enzyme with some... 

The characterisation of the complexation between flavocytochrome b2 and cytochrome c has been the subject of many studies (see for example Short et al., 1998 Daff et al., 1996b and CapeillEre-Blandin, 1995). Work on the anomala flavocytochrome b2, for which there is no crystal structure, led to the conclusions that the cytochrome c binding site involved both the flavodehydrogenase and cytochrome domains (CapeillEre-Blandin and Albani, 1987) and that the complex was stabilised by electrostatic interactions (CapeillEre-Blandin, 1982). It is clear that similar conclusions hold true for the S. cerevisiae enzyme (Daff et al., 1996b) for which the crystal... 

The model proposed by Short et al. (1998) has been rigorously tested by site-directed mutagenesis and kinetic analysis of the mutant enzymes. It has been shown to be consistent with all of the results from these studies and therefore it may indeed represent the physiological complex formed between flavocytochrome 4 2 cytochrome c. 

There are a number of factors which make flavocytochrome b2 an ideal model system for studying both intra- and inter-molecular electron transfer. Reasons include (i) the fact that it has been expressed at a high level in E. coli (Black et al., 1989) and is soluble and easily obtained (ii) crystal structures of the native (Xia and Mathews, 1990) and recombinant (Tegoni and Cambillau, 1994) enzymes are available (iii) a hypothetical structure for the flavocytochrome 4 2 cytochrome c complex has been proposed (Short et al., 1998) (iv) many mutant forms of the enzyme have been constructed (v) there is a wealth of data on the mechanism of action of the enzyme (Chapman et ah, 1991 Lederer, 1991). 

Lindqvist, Y., Br%ondEn, C-L, Mathews, E. S., and Lederer, E., 1991, Spinach glycolate oxidase and yeast flavocytochrome foj sre structurally homologous and evolutionarily related enzymes wioth distinctly different function and EMN binding, J. Biol. Chem. 266 3198n3207. 

Fig. 2. (A) A schematic diagram of equine Cyt c from the front of the heme crevice. The approximate positions of the /8-carbons of the lysine residues are indicated by closed and dashed circles for residues located toward the front and back of the molecule, respectively. Differential chemical modification indicates that some residues are protected by both flavocytochrome c-552 and mitochondrial redox partners (cross-hatched), or only by flavocytochrome c-552 (hatched), or only by mitochondrial enzymes (stippled). (B) Comparison of reactivity ratios (R) obtained by differential chemical modification of equine Cyt c in the presence and absence of flavocytochrome c-552 (filled bars), mitochondrial Cyt foe, complex (left open bar) and mitochondrial Cyt c oxidase (right open bar). Data for mitochondrial redox partners are from Ref. 98. In the case of the mitochondrial redox partners, R values for lysines 55, 72 and 99 are average values for lysines 53-t-55, 72+73 and 99+100. The R values represent, after a series of corrections, the ratio of acetylation of a specific lysine residue in free Cyt c to the acetylation of the same residue in the Cyt c flavocytochrome c-552 complex. The larger the R value, the greater the extent of protection against acetylation.

In 1915, Harden and Norris observed that dried yeast, when mixed with lactic acid, reduced methylene blue and formed pyruvic acid 4). Thirteen years later Bernheim prepared an extract from acetone-dried baker s yeast, which had lactate dehydrogenase activity (5). Bach and co-workers demonstrated that the lactate dehydrogenase activity was associated with a 6-type cytochrome, which they named cytochrome 62 (6). In 1954, the enzyme was crystallized, enabling the preparation of pure material and the identification of flavin mononucleotide as a second prosthetic group (2). Since then, significant advances have been made in the analysis of the structure and function of the enzyme. Much of the earlier work on flavocytochrome 62 has already been summarized in previous review articles (7-10). In this article we shall describe recent developments in the study of this enzyme, ranging fi om kinetic, spectroscopic, and structural data to the impact of recombinant DNA technology. 

Purification procedures have been reported for flavocytochromes 62 from the yeasts Saccharomyces cerevisiae (baker s yeast) and Han-senula anomala. Each of these enzymes has subsequently been subjected to extensive characterization. 

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