Diiron centers

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... 

Hydroxylase in the mixed-valent Fe(II)Fe(III) oxidation state (Hmv) is readily accessible by one-electron reduction of the dinuclear center. Mossbauer data indicate the presence of one Fe(III) and one Fe(II) (39). Hmv has a rhombic EPR signal with gav = 1.83 (27, 37) and J -30 cm1 (38,39), properties characteristic of other mixed-valent nonheme carboxylate-bridged diiron centers such as that in semimet hemerythrin (J = -15 cm-1) (32). ENDOR spectroscopic studies of Hmv... 

Fig. 1. Structure of the active site of H0I as determined by x-ray crystallography (41). Broken lines are used to designate hydrogen bonding of the water molecule to Glu 114 and Glu 243, as well as the semibridging interaction of Glu 144 with the diiron center.

Determination of the reduction potentials of the diiron center in the hydroxylase, shown in Eq. (2), and investigation of the effects of the other two MMO components on those potentials, have revealed different behavior for the two organisms (Table II). 

As indicated by the negative shifts in the reduction potentials of Hox, protein B can interact with the diiron center in Hmv from both MMO systems (63, 64). Consistent with this interpretation are EPR studies of Hmv from both organisms which indicate that, in the presence of protein B, the EPR signal moves from gav 1.83 to gav 1.75 (48, 66). 

Davydov, R., Kuprin, S. Graslund, A., and Ehrenberg, A. 1994. Electron paramagnetic resonance study of the mixed-valent diiron center in Escherichia coli ribonucleotide reductase produced by reduction of radical-free protein R2 at 77 K. Journal of the American Chemical Society 116 11120-11128. 

Figure 16-20 (A) The active site of hemerythrin showing the two iron atoms (green) and their ligands which include the (X oxo bridge and two bridging car-boxylate groups. From Lukat et al.193 The active site is between four parallel helices as shown in Fig. 2-22. (B) Stereoscopic view of the backbone structure of a A9 stearoyl-acyl carrier protein desaturase which also contains a diiron center.

Notice the nine-antiparallel-helix bundle. The diiron center is between four helices as in hemerythrin. (C) Stereoscopic view of the diiron center of the desaturase.333 (B) and (C) courtesy of Ylva Lindqvist. 

Each polypeptide chain of the P2 dimer or R2 protein contains a diiron center which serves as a free radical generator 354a b/C A few bacteria utilize a dimanganese center.355 Oxygenation of this center is linked to the uptake of both a proton and an electron and to the removal of a hydrogen atom from the ring of tyrosine 166 to form HzO and an organic radical (Eq. 16-23) 356-360... 

Figure 16-21 (A) Scheme showing the diiron center of the R2 subunit of E. coli ribonucleotide reductase. Included are the side chains of tyrosine 122, which loses an electron to form a radical, and of histidine 118, aspartate 237, and tryptophan 48. These side chains provide a pathway for radical transfer to the R1 subunit where the chain continues to tyrosines 738 and 737 and cysteine 429.354a c From Andersson et al.35ic (B) Schematic drawing of the active site region of the E. coli class IH ribonucleotide reductase with a plausible position for a model-built substrate molecule. Redrawn from Lenz and Giese373 with permission.

The origin of ricinoleic acid, an abundant constitu-tuent of castor beans, is also shown in Fig. 21-2. It is formed by an oleate hydroxylase that has an amino acid sequence similar to those of oleate desaturases.113 Both hydroxylation and desaturation are reactions catalyzed by diiron centers.114 Other fatty acid hydroxylases act on the alpha115 and the omega positions. The latter are members of the cytochrome P450 family.116 117... 

The corresponding high-valent intermediate in the assembly of the di-iron(III) center-tyrosyl radical cofactor of RNR R2 has also been identified by Stubbe and coworkers and designated as X [86,89], This intermediate decays to the (p,-oxo)diiron(III) form at a rate commensurate with the appearance of the tyrosyl radical. Intermediate X, formally Fe(III)Fe(IV), exhibits an isotropic S = 1/2 spin EPR signal near g = 2, which is split by the introduction of 57Fe and broadened by 1702 in the assembly reaction. These observations as well as Mossbauer results show that the unpaired spin must be associated with the diiron center [88,89],... 

The oxidative mechanism of RNR R2 differs from that of MMOH in requiring an additional electron, since Tyrl22 provides only a single electron. This electron is needed to convert P to X. It has been shown that external reductants such as excess Fe(II) or ascorbate can provide this electron in in vitro reconstitution reactions [87,97], Since the diiron site is buried 10 A below the protein surface, a long-range electron transfer pathway is required to deliver this extra electron to the diiron center. Such a pathway involving a number of amino acid residues has been proposed from examining the crystal structure of R2 [98],... 

Often we do not initially know whether a spectrum like that of Figure 2.5a results from high-spin Fe3 +. For instance, a low-spin Fe2 + (S = 0) complex may yield AEQ and 5 values similar to those of the spin-coupled diiron center. In that case, addition of reductants or oxidants may settle the problem. 

Evidence for a Diiron Center in Particulate Methane Monooxygenase... 

Martinho, M. Choi, D. W. Dispirito, A. A. Antholine, W. E. Semrau, J. D. Miinck, E. Mossbauer studies of the membrane-associated methane monooxygenase from Methylococcus capsulatus bath evidence for a diiron center. J. Am. Chem. Soc. 2007, 729(51), 15783-15785. 

Conformational changes in diiron center of stearoyl-acyl carrier protein desaturase caused by substrate binding have been probed by EPR and proton ENDOR of cryoreduced diferric protein.80 EPR spectra of the one-electron reduced Fe(III)Fe(II)... 

The latter compound attracts special interest because it forms more rapidly in the absence of substrates (k = 1.2 s 1) than it autodecays (k = 0.05 s 1) and, therefore, can be directly investigated by physicochemical methods. The Mossbauer spectrum of compound Q from M. trichosporium indicates that the diiron center consists of two high-spin antiferromagnetically-coupled iron atoms, each in the Fe(IV) state bridged by oxygen atom. Compound Q reacts very quickly with methane and other substrates with the formation of compound T. The latter releases a product and is transformed to diferric MMOH. 

Willems, J-P., Valentine, A.M., Gurbiel, R., Lippard, S.J., and Hoffman, B.M. (1998) Small molecule binding to the mixed-valent diiron center of methane monooxygenase hydroxylase from Methylococcus capsulatus... 

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