Substrate hydroxylation

Under conditions of copper deficiency, some methanotrophs can express a cytosolic, soluble form of MMO (sMMO) (20-23), the properties of which form the focus of the present review. The sMMO system comprises three separate protein components which have all been purified to homogeneity (24,25). The hydroxylase component, a 251 kD protein, contains two copies each of three subunits in an a 82y2 configuration. The a subunit of the hydroxylase houses the dinuclear iron center (26) responsible for dioxygen activation and for substrate hydroxylation (27). The 38.6 kD reductase contains flavin adenine dinucleotide (FAD) and Fe2S2 cofactors (28), which enable it to relay electrons from reduced nicotinamide adenine dinucleotide (NADH) to the diiron center in the... 

Figure 5 presents some possible mechanisms for substrate hydroxylation by intermediate Q additional possibilities are discussed elsewhere (59). Mechanisms A-C assume that this species is a diiron(IV) oxo... 

Since the two-spin state forms can lead to different products, the products obtained will be a mixture that reflects the initial fractionation of the reaction between the two-spin states. The fractionation in turn is a reflection of the interplay and the probability of cross-over between the two-spin states (8). Thus, the two-state reactivity paradigm resolves the dilemma of whether a radical recombination or a direct insertion mechanism governs cytochrome P450-catalyzed hydroxylation actually they are both involved and the degree to which either is expressed depends upon the specific substrate hydroxylated and the specific enzyme. 

Sphingomonas sp. HXN-200 was also able to accept a six-member ring substrate. Hydroxylation of A/-benzyl- and A/-/ert-butoxycarbonyl-2-piperidinone gave the corresponding (/J)-4-hydroxy-piperidin-2-ones in 31% and 68% ee, respectively (Figure 15.4). This provides a simple synthesis of such types of useful synthons. 

Myoglobin in many respects is the prototypical example of the larger family of heme containing proteins and enzymes that vary in function from the relatively simple process of reversible binding of an electron to the activation of dioxygen for substrate hydroxylation. The relationship between members of this family of proteins is not based simply on structural similarities but on similarities in chemical reactivity as well. As the structure of myoglobin is relatively simple compared to other heme proteins and as it was the first for which the three-dimensional... 

In subsequent work, Hamberg has reported that anerobic oxidation of lineolic acid by cumene hydroperoxide catalyzed hy sperm whale Mb results in formation of five products, the two major products being ll(i ,S)-hydroylinoleic acid (29% 5deld) and ( )cis-9,10-epoxy-(12Z)-octadecenoic acid (16%) (235). In this work, it was proposed that the second oxidizing equivalent required for substrate hydroxylation was probably provided by a protein-centered radical. 

An intriguing puzzle in NOS catalysis is the precise role of H4B. The traditional function of H4B is in aromatic amino acid metabolism where H4B directly participates in the hydroxylation reaction via a nonheme iron. However, the NOS pterin site has no similarity to the pterin site in the hydroxylases, nor does NOS have a nonheme iron to assist pterin in substrate hydroxylation as in the amino acid hydroxylases 111). NOS more closely resembles pterin-containing enz5unes that have a redox function 81). In particular, N3 and the 03 amino group form H-bonds with either GIu or Asp residues in a series of pterin enzymes 112-116) similar to NOS, except that NOS utilizes the heme propionate (Fig. 6). 

Once the oxy complex is formed, a second electron transfer to the HO heme effectively reduces the oxy complex to the peroxide level. From this point many heme enzymes catalyze the heterolytic fission of the peroxide 0-0 bond, leaving behind the well known oxyferryl center, (Fe-0) +, characteristic of peroxidase compound 1 and similar to the active hydroxylating intermediate thought to operate in P450s. However, in HO the active oxidizing intermediate is peroxide. Peracids that form the (Fe-0) + intermediate do not support the HO reaction, whereas H2O2 addition to Fe + HO does support substrate hydroxylation 187, 188). EPR and ENDOR spectroscopy have been used to analyze the cryo-genically reduced oxy-HO complex 189). In these studies reduction of... 

Raag, R. and Poulos, T. L. (1991) Crystal structures of cytochrome complexed with camphane, thiocamphor and adamantane factors controlling P450 substrate hydroxylation. Biochemistry 30, 2674-2684. 

A. Rivera-Sagredo, F. J. Canada, O. Nieto, J. Jimenez-Barbero, and M. Martin-Lomas, Substrate specificity of small-intestinal lactase Assessment of the role of the substrate hydroxyl groups, Eur. J. Biochem., 209 (1992) 415 122. 

In this case the ferric iron site seems to activate the substrate. Our generalization of this model is shown in Fig. 7. The iron binds the substrate and could serve, as a Lewis acid, to facilitate ketolization of either the bidentate or monodentate substrate. The hydroxide (water) ligand may be more significant than simply a placeholder ligand to be displaced by substrate. Since both substrate hydroxyls must lose their... 

Fig. 19. Calculated free energy for the FeIV=0-mediated substrate hydroxylation.

Scheme 5. The electronic rearrangements of the aromatic amino acid substrate hydroxylation.

The above criteria apply in the case of isolated hydroxyl groups but when additional polar substituents are placed in the vicinity of the substrate hydroxyl the oxidation rate can be expected to change. Allylic hydroxyls are generally oxidized more rapidly than their saturated counterparts. Burstein and Rin-gold have studied the chromic acid oxidation of steroidal allylic alcohols in some detail and have found that the quasi-equatorial 3)3-isomer is oxidized more... 

Chloramphenicol and secobarbital exhibit properties similar to those of tienilic acid, but they have not been studied in humans (11). Oxidative dechlorination of chloramphenicol with formation of reactive acyl chlorides appears to be an important metabolic pathway for irreversible inhibition of CYP. Chloramphenicol binds to CYP, and subsequent substrate hydroxylation and product release are not impaired. The inhibition of CYP oxidation and the inhibition of endogenous NADPH oxidase activity suggest that some modification of the CYP has taken place, which inhibits its ability to accept electrons from the CYP reductase (11). Secobarbital completely inactivates rat CYP2B1 functionally, with partial loss of the heme chromophore. Isolation of the N-alkylated secobarbital heme adduct and the modified CYP2B1 protein revealed that the metabolite partitioned between heme N-alkylation, CYP2B1 protein modification, and epoxidation. A small fraction of the prosthetic heme modifies the protein and contributes to the CYP2B1 inactivation (12). 

Methane and other substrate hydroxylation by dioxygen occurs with the participation of all three components of the enzyme MMOH, MMOR and MMOB (Feig and Lippard, 1994 Wallar and Lipscomb, 1996 and references therein). The redox potential of the transition [Fe(III)-Fe(III)] -> [Fe(II)-Fe(II)] is E0 = 0.048 V in the MMOH resting state and changes into -0.084, +0.097 and +-0.100 V after the addition of MMOB, MMOR and (MMOB +- MMOR), respectively (Waller and Lipscomb, 1996). 

Among other discussed concepts concerning the MMO substrate hydroxylation in the compound Q active site, the following suggested mechanisms should be mentioned. 

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