MMOH Bath

Interestingly, the detailed structure of the oxidized binuclear iron cluster environment depends to some extent on the temperature at whieh the data was collected and on the origin of the MMOH. The most similar structures, and those most likely to be representative of the enzyme in vivo, are derived from flash frozen MMOH Bath crystals (nl60 C) and MMOH... 

Physical Properties of MMOH from M. trichosporium OB3b and M. capsulatus (Bath)... 

Figure 2.4 X-ray crystal structures of different forms of MMOH from M. capsulatus (Bath) coordinates taken from PDB files 1FYZ (a) [59] and 1FZ6 (b) [60]. Coordinative bonds are represented bysolid linesand hydrogen bondsor weak bonds by dotted lines. Only the amino acid side-chains of the active site and coordinated aqua/hydroxido/methoxido ligands are shown. Hydrogen atoms have been omitted for clarity.

Scheme 2.7 The catalytic cycle of MMOH based on experimental and computational results [41]. The amino acids are numbered according to the sequence of M. capsulatus (Bath).

The crystallographic structures of MMOH from both microorganisms, Methylococcus capsulatus (Bath) and Methylosynus trichosporium OB3b have been... 

FIGURE 4. Backbone X-ray crystal structure of MMOH isolated from M. trichosporium OB3b. The cylinders represent helicies. The arrow points toward one of the two active sites in the (aPY)2 structure. (Elango et al, 1997). The structure of the M. capsulatus Bath MMOH is essentially identical (Rosenzweig et al, 1993). 

The addition of MMOH does not affect the rates of electron transfer between NADH and oxidized FAD. However, in the MMO Bath system, the presenee of MMOH results in less semiquinone formation, and the reduction of the [Fe2S2] cluster is delayed. This suggests that when the iron-sulfur cluster of MMOR is reduced, intermolecular electron transfer to the diiron center of MMOH is very fast. Subsequently, the second electron is... 

FIGURE 7. Electron transfer pathway and rates from NADH through MMOR to MMOH isolated from M. capsidatus Bath. (From Gassner et al., 1999). 

Fig. 1. Diferric iron clusters form hemer3fthrin, ribonucleotide reductase R2 subunit, and methane monooxygenase hydroxylase. The figure was made with the RasMol 2.0 program, and the protein coordinates as PDB files were obtained from Brookhaven Protein Data Bank. Only the amino acids (histidines, green carboxylates, black oxygen, red nitrogen, yellow acetate, blue iron, violet) coordinated to the iron cluster are shown, coordinated waters are not indicated. The first subunit containing the cluster is shown. Diferric Hr is from sipunculid worm Themiste dyscritra). The RNR-R2 is from E. coli. The MMOH is from Methvlococcus caosulatus (Bath).

Figure 1 Ribbon diagram of four-helix bundle of MMOH from M. capsulatus (Bath) with the diiron active site depicted in ball-and-stick form (left). Ball-and-stick diagram of the MMOH, RNR-R2, and A9D active...

MMOH from M. capsulatus (Bath) has a molecular weight of about 210 kDa and is composed of three subunits of Mr = 54, 42, and 17 kDa. The subunits are present in stoichiometric amounts forming an p2 Y2 arrangement in the native protein. The MMOH has a substrate binding site, as suggested by means of ESR spectroscopy [30]. The MMOH is a competent catalyst by itself, capable of oxidizing a variety of substrates... 

Spectroscopic studies have demonstrated that the MMOHs have the stable diiron centers of diferric Fe(III)Fe(III), mixed-valent Fe(II)Fe(III), and diferrous Fe(II)Fe(II) redox states [33]. Although the electronic absorption spectrum of the MMOH from M. capsulatus (Bath) shows an absorption peak at 280 nm due to the protein, there is no significant optical features that can be attributed to the iron prosthetic group. The Mdssbauer Fe enriched spectrum of the isolated MMOH from M. trichosporium OB3b shows two quadrupole doublets of equal area. Their parameters at 4.2 K are AEq =1.16 mm/s and the isomer shift 5 = 0.51 mm/s for site 1 and AEq = 0.87 mm/s and the isomer shift 6 = 0.50 mm/s for site 2 [34]. The Mdssbauer spectrum of the isolated MMOH from Af. capsulatus (Bath) at 80 K shows a quadrupole doublet having AEq = 1.05 mm/s and the isomer shift 6 of 0.50 mm/s [35]. These spectra show that the isolated MMOH is diferric form. 

This weak interaction indicates that the diferric ions are bridged by a hydroxide, alkoxide, or monodentate carboxylate. The EXAFS data for the diferric form of MMOH from M.capsulatus (Bath) indicate that the Fe Fe distance and the average distance of the Fe-0/N bonds are 3.42 and 2.04 A, respectively [35, 37]. A short Fe-0 bond is not observed in the EXAFS experiments, indicating that the diferric center does not have a i-oxo bridge. 

The redox properties of MMOHs from M. capsulatus (Bath) and M. trichosporium OB3b were reported. The measurements were carried out by potentiometric titrations which were combined with ESR spin quantitation of the mixed valent MMOHs (Fe(II)Fe(III)) formed in the electrochemical reaction [33, 36, 49]. The data of the redox potentials of the MMOHs are shown in Table 2. 

The X-ray crystal structure of MMOH from M. capsulatus (Bath) has been determined by Lippard et al at 2.2 A resolution [52, 53]. The X-ray diffraction data were collected at 4 The enzyme is a relatively flat molecule with approximate dimensions 60 x 100 x 200 A. The arrangement of the subunits is similar to the schematic drawing in Scheme 1. The two a-subunits contact one another and so do two P-subunits with more extensive contacts. The y-subunit lies on the outer edges of the molecule at the interface of the a-and p-subunit. The two apy protomers are related by a non-crystallographic two fold symmetry and form a heart-shaped molecule. There is a large canyon at the interface between the two aPy protomers. The secondary structure of the three subunits is primarily helical, with one small region of p-structure in the a-subunit. The a-subunit involves 18 helices and two P-hairpin motifs. The P-subunit has 12 helices. The y-... 

Lippard et al detected new intermediates, compounds L and Q in the catalytic cycle of MMOH from M. capsulatus (Bath) [65]. These are similar to the compounds P and Q, respectively, for MMOH from M. trichosporium OB3b. The intermediates were trapped by applying the same method as Lipscomb et al [61, 62]. After addition of dioxygen gas to the diferrous MMOH, the compound L was detected from the sample of 155 ms interval, and the compound Q was detected from the sample of 3 s interval. Resonance Raman spectrum of the compound L shows that the compound L is a diiron peroxo complex [66]. The detailed analysis of the Mossbauer spectrum of the compound L shows that the compound L has a synmietrical structure and suggests that the peroxo ligand of compound L coordinates in the I-T t or the I-T ti binding mode. 

Figure 1. Overall structure of MMOH from Methylococcus ccpsulatus (Bath) (PDB code 1FZ2).

---