Diferric MMOH

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. 

A third eatalytie system consists of chemically or electrochemically reduced MMOH, substrate and O2(Fox eta/., 1989 Froland eta/., 1992). In this system, diferrous MMOH reacts with O2 and turns over a single time to yield the expeeted products. This system is ideally constituted to search for intermediates in the reaction cycle and to determine the rate constants for the formation and deeay of these intermediates. 

FIGURE 8. Postulated mechanism for MMO. The inner cycle are postulated intermediates in the catalytic cycle (only the binuclear iron cluster of the MMOH component is shown). The outer cycle represents the intermediates detected during a single turnover beginning with diferrous MMOH and ending with diferric MMOH. The rate constants shown are for 4 C and pH 7.7. The rate shown for the substrate reaction RH with Q is that for methane. The alignment of the two cycles shows the postulated structures for the intermediates. 

The rate of P formation was decreased when the pH was increased, whereas the rate of diferrous decay was not affected (Lee and Lipscomb, 1999). The different effects of pH indicate that there must be an intermediate between diferrous MMOH and P. 

It seems from product distribution studies that even after dissociation of the MMOB from the diferrous MMOH, the active site in MMOH remembers that it had just been in complex with MMOB. This is presumably because MMOB induces a metastable (on the time scale of catalysis) conformation of the MMOH (129). The effect is observed as a hysteresis effect upon titration with MMOB to the diferrous MMOH (see Fig. 13 for a model), where a ratio 0.1 of MMOB/(MMOH active site) could induce a complete metastable conformation of MMOH. This could be observed by a drastic change in product distribution, and no further change in product distribution was observed in the presence of higher ratios of MMOB/MMOH. The model was corroborated by the low ratio of MMOB/MMOH needed to induce the MMOB-dependent changes of the diferrous MMOH EPR signal (129). The affinity between MMOB and MMOH depends on the redox state of MMOH. This explains... 

Fig. 13. Hypothetical scheme to explain semistable conformation of diferrous MMOH after interaction with MMOB. MMOB (B) can rapidly change the diferrous MMOH (H ) conformation. H keeps this changed conformation for some time after its contact with MMOB, so that MMOB at a particular time needs to bind to only a small proportion of MMOH during the rapid catalysis. When MMOH is in the diferric (H°) state, it can form a one-to-one complex with MMOB. Adapted from (.129).

The unique g = 16 EPR signal of diferrous MMOH allowed its reaction with O2 to be followed using rapid freeze-quench procedures in conjunction with EPR... 

Figure 19-2 Intermediates observed in the single turnover cycle of diferrous MMOH in the presence of MMOB after rapid mixing with O2 and substrate containing solution. The rates shown are for the reaction in pH 7.7 buffer at 4 °C. The intermediate R is proposed from chemical rather than kinetic studies. The rate of the reaction of Q with substrates depends on the specific substrate used and appears to be second order overall, but for comparison, a typical pseudo first-order rate constant is given for the reaction of Q with nitrobenzene at a concentration of 2.S mM.

MMOH. T decays to diferric MMOH by releasing product in the overall rate-limiting step of catalysis the rate of this step is equal to the turnover number in the steady state turnover of nitrobenzene. 

Figure 19-6 Time dependence and yield of propene oxide from propene during a single turnover of diferrous MMOH with and without MMOR and MMOB. ( ) Diferrous MMOH alone. ( ) Plus equimolar MMOB. (a) Plus equimolar MMOB and MMOR.

The complexes between MMOH and the other components also have other more subtle effects. As illustrated in Figure 19-6, a single turnover of diferrous MMOH to form product in the absence of MMOB and MMOR gives about 40% of the theoretical yield [26, 27]. This is actually a very good yield considering that the reduced protein must be exposed to O2 for more than S minutes in order to fully turn over under these conditions. In the presence of MMOB, the yield increases to 80%. This appears to be due to both the increased rate of reaction with O2 and also to an increase in the rate of P to Q conversion. It seems likely that if P is not rapidly con-... 

Lipscomb et al detected three new intermediates in the catalytic cycle of sMMO isolated from M. trichosporium OB3b [61, 62] At 4 °C, the diferrous MMOH reacts with O2 in the presence of a 2-fold excess amount of protein B. The reaction has been followed by means of ESR and electronic absorption spectroscopies. The g = 16 ESR signal characteristic of the diferrous MMOH disappears at an apparent first order rate of 22 5 s" A new species having. ax and 430 nm (emax- 7500 M cm for each band) is generated with an average first order rate constant of 1 0.1 s" and then decayed at 0.05 0.01 s This intermediate was named compound Q. Since the formation rate constant of the compound Q is much similar than the apparent decay rate constant of diferrous state, the formation of another intermediate before the compound Q is assumed. 

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. 

Diferric MMOH. Fully oxidized, diferric MMOH is diamagnetic (Stat = 0) at low temperatures, indicating the presence of two antiferromagnetically coupled high-spin Fe ions (Spei = S Fe2= 5/2) [70], Using parallel mode X-band continuous-... 

An alternative method to generate the heterovalent Fe -Fe form of MMOH involves the use of y-irradiation at low temperatures (77 K radiolytic reduction) [87,88]. Since the one-electron reduction is carried out at low temperature, structural changes to the MMOH active site are anticipated to be minimal, essentially leaving the conformation of the diferric MMOH cluster virtually intact [87]. Thus, radiolytic reduction provides an avenue to study by EPR (i) the geometric structure... 

---