Redox enzymes flavocytochrome

One of a few cases in which electron transfer of redox enzymes is expressed directly and reversibly at an electrode is concerned with p-cresol methylhydroxylase (PCMH). This is a flavocytochrome c enzyme of 115 kDa, which catalyzes the oxidative hydroxylation of p-cresol to p-hydroxybenzyl alcohol and subsequently to p-hydroxyben-zaldehyde. The structure of PCMH has recently been determined (56) at 3 A resolution. It is an a2 2 tetramer, with one subunit containing a covalently bound FAD and the other containing a c-type heme group. 

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. 

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. 

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.. 

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.

Yeast mitochondrial flavocytochrome 2 (lactate cytochrome c oxido-reductase) catalyzes the transfer of electrons from L-lactate to various acceptors, cytochrome c being the physiological acceptor. The protoheme and flavin mononucleotide, because of their higher redox potential, can both be reduced completely by L-lactate the enzyme accepts a total of three electrons per protomer, which amounts to twelve electrons for the stable active tetramer. Both types of prosthetic group are quantitatively reoxidized by external acceptors. 

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