P-Cresol methylhydroxylase

There are five classes of flavin-binding structural folds presented in Table 1 that are identified by the prototype protein in which they were first discovered. These are flavodoxin (FDX), ferredoxin reductase (FNR), triosephosphate isomerase (TIM), glutathione reductase (GR) and p-cresol methylhydroxylase (PCMH). The topologies of four of these five domains are shown in Figure 2. There are also four classes of primary acceptor/donor domain folds that are identified by the prototype protein where they were first discovered. They are cytochrome P450BMP (BMP), cytochrome b5 (CYTB5), cytochrome c (CYTC) and the 2Fe-2S plant-type ferredoxin (FDN). 

FIGURE 8. stereo diagram of p-cresol methylhydroxylase. The flavoprotein subunit is on the left and the cytochrome subunit is on the right. The flavin-binding domain of the flavoprotein subunit is on the bottom and the catalytic domain is on the top. Skeletal models of the heme and FAD prosthetic groups are also shown. 

Bhattacharyya, A., Tollin, G., Mclntire, W. S., and Singer, T. P., 1985, Laser-flash-photolysis studies of p-cresol methylhydroxylase. Electron-transfer properties of the flavin and haem components, Biochem. J. 228 337n345. 

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. 

Kim, J., Fuller, J. H. Cecchini, G., and Mclntire, W. S. 1994, Cloning, sequencing, and expression of the structural genes for the cytochrome and flavoprotein subunits of p-cresol methylhydroxylase from two strains of Pseudomonas putida. J. Bact. 176 634996361. 

Mclntire, W., Edmondson, D. E., Hopper, D. J., and Singer, T. P., 1981, 8 alpha-(0-tyrosyl)flavin adenine dinucleotide, the prosthetic group of bacterial p-cresol methylhydroxylase. Biochemistry 20 3068n3075. 

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. 

The catalytic current was obtained using a scan rate of 10 mV sec , at pH 7.0, in the presence of p-cresol methylhydroxylase (1 iJiM) and p-cresol (2 mM) at an edge-plane graphite electrode. Numbers in parentheses refer to structures shown in Fig. 8. 

Fic 8. Structures of some of the cationic molecules used as promoters for p-cresol methylhydroxylase electrochemistry. 

Fig. 9. Direct electrochemistry of p-cresol methylhydroxylase. (a) Response at an edge-plane graphite electrode in 10 toM KCl/10 toM HEPES (pH 7.4) buffer containing 10 toM spermine. Scan rate 5 mV sec", (b) Response upon addition of enzyme to —30 ItM. (c) A repeat of (b) at reduced sensitivity, (d) Catalytic response upon addition of p-cresol (3 roM) to solution.

Mclntire, W., D.J. Hopper, and T.P. Singer. 1985. p-Cresol methylhydroxylase. Assay and general properties. Biochem. J. 228 325-335. 

Hopper, D.J. 1988. Properties of p-cresol methylhydroxylases. In Microbial Metabolism and the Carbon Cycle pp. 247-258 (Eds. S.R. Hagedorn, R.S. Hanson, and D.A. Kunz). Harwood Academic Publishers, Chur, Switzerland. 

The application of direct electrochemistry of small redox proteins is not restricted to cytochrome c. For example, the hydroxylation of aromatic compounds was possible by promoted electron transfer from p-cresol methylhydroxylase (a monooxygenase from Pseudomonas putida) to a modified gold electrode [87] via the blue copper protein azurin. All these results prove that well-oriented non-covalent binding of redox proteins on appropriate electrode surfaces increases the probability of fast electron transfer, a prerequisite for unmediated biosensors. Although direct electron-transfer reactions based on small redox proteins and modified electrode surfaces are not extensively used in amperometric biosensors, the understanding of possible electron-transfer mechanisms is important for systems with proteins bearing catalytic activity. 

Fig. 2. Graph showing how voltammetric peak current is expected to diminish with increasing molecular weight in the case of redox-active spherical molecules diffusing to a planar electrode. PCMH = p-cresol-methylhydroxylase (see Sect 7.3)...

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