Front Matter

structure). The redox potential is thereby increased from – 300 mV to – 173 mV,

facilitating the oxidation or direct oxygenation of the iron center via the shunt path-

way ( 2 R 6 ) by strong oxidants such as peroxides, periodate or peracids (Coon et al.,

1996; Karuzina and Archakov, 1994; Joo et al., 1999a). Under physiological con-

ditions, a one-electron reduction occurs, resulting in the formation of a high-spin

FeIIcenter (S¼2) 3 (Poulos and Raag, 1992). This configuration with four unpaired

electrons is well prepared to bind triplet oxygen. The low-spin dioxy-iron (III) com-

plex 4 was isolated and characterized by cryocrystallography (Schlichting et al.,

2000) revealing the iron atom in-plane to the porphyrin system. The next step of

the reaction cycle, a further one-electron reduction, produces a very unstable per-

oxo-iron complex 5. The structure of the end-on or site-on coordinated dioxygen

species is uncharacterized until now (Schlichting et al., 2000). For the cleavage

of the iron-bound dioxygen, a two-proton shuttle is realized, involving water mo-

lecules and amino acid residues Asp 251 , Asn 255 and Thr 252 (Gerber and Sligar,

1994; Vidakovic et al., 1998) of P450cam in a ‘protein-solvent hydrogen-bonding

network’ (Schlichting et al., 2000). The oxy-ferryl species 7 , produced with simul-

taneous formation of water, has a rather planar heme, having the iron slightly above

the plane and a short iron-to-oxygen distance suggesting an Fe¼O bond. The oxi-

dation state of the iron in this species and the electronic state of the heme could not be

determined from the crystal structures. The removal of water allows the camphor

molecule to move toward the heme, to be hydroxylated via a P450cam – substrate

complex (Schlichting et al., 2000) to 5-hydroxycamphor and thereby regenerating

the low-spin FeIII 1 center. The postulated rebound mechanism (Ortiz de Montellano,

1995; Filatov et al., 1999) is under debate due to recent mechanistic studies (Toy et

al., 1998) using ultrafast radical clocks to probe the presence of a free radical, that

suggests a concerted oxene-insertion mechanism.

18.4 Fatty Acid-hydroxylating P450s Monooxygenases

The fatty acid-hydroxylating P450 enzymes can be divided – depending on the

hydroxylation products – into terminal and subterminal fatty acid hydroxylases

(Figure 2).

The hydroxylation of a nonactivated –C–H bond of a terminal –CH 3 group is

thermodynamically 3 kcal mol–1(Ortiz de Montellano et al., 1992) inferior to

the hydroxylation of a secondary –CH 2 -group. It requests higher steric demands

toward the P450 enzyme with respect to substrate fixation, orientation and control

of the hydroxylation cycle. The product profile of terminal and subterminal fatty acid

hydroxylases clearly reflects these differences between the P450 species. The cyto-

chrome P450 fatty acidx-hydroxylases are a distinct family (CYP4) (Ortiz de Mon-

tellano et al., 1992) of cytochrome P450 enzymes, that exhibit a high preference for

the hydroxylation of the terminal –CH 3 of saturated and unsaturated fatty acids,

including compounds such as prostacyclins, thromboxanes and prostaglandins

(Kupfer, 1980). Rather little is known about the mechanism ofx-hydroxylation

due to a lack of crystal structures. Nevertheless, it is likely thatx-hydroxylating

P450 enzymes achieve their regioselectivity by steric factors through nonbonding

398 18 Fatty Acid Hydroxylations using P450 Monooxygenases