Abduction of iron(III) from the soluble methane monooxygenase hydroxylase and reconstitution of the binuclear site with iron and manganese

Abstract

The apo-form of the soluble methane monooxygenase hydroxylase from Methylococcus capsulatus (Bath) was prepared via chelation of iron(II1) with 3,4-dihydroxybenzaldehyde. The apohydroxylase was reconstituted by the anaerobic addition of Fe(I1) followed by air oxidation. The enzyme thus prepared regained 85-90% of its original catalytic activity. The incorporation of two manganese(I1) ions/mol of apohydroxylase was monitored by EPR spectroscopy. The Mn(I1) ions occupy the native diiron active site and remain in the +2 oxidation state. The EPR data suggest strong coupling between the two Mn(I1) ions and retention of the p-hydroxo (alkoxo) bridge. The results of this study indicate that the M. capsulutus (Bath) hydroxylase contains a single diiron site.Methane monooxygenase (MMO) efficiently catalyses the conversion of methane to methanol at ambient temperature and pressure. There are two forms of the enzyme, soluble (sMMO) or membrane-bound particulate (pMMO), the occurrence of which depends on the Cu2+ : biomass ratio under which the cells are grown [l]. Soluble methane monooxygenase has been the more studied and is a three-component enzyme system as isolated from several different organisms [2-71. Electrons from NADH are passed through the FAD and Fe2S, centers of the reductase to the hydroxylase [2, 81. The third component, protein B, contains no metal ions nor cofactors and is believed to have a regulatory function [9-111.The hydroxylase has been shown to be the component within which oxidation of methane and a variety of other substrates occurs 141. The active site is a p-hydroxo-bridged or -alkoxo-bridged diiron cluster [12-141 similar to those found in other nonheme iron-oxo proteins like the R2 subunit of ribonucleotide reductase, hemerythrin, purple acid phosphatases and rubrcrythrin [ 15 -191. The diiron centers perform very different functions in each of these metalloproteins. In R2 a stable tyrosyl radical is generated, in hemerythrin oxygen is bound reversibly, purple acid phosphatases catalyse the hydrolysis of phosphate esters and sMMO catalyses the oxidation of many alkane, alkene, heterocyclic and aromatic compounds. The X-ray crystal structures of the R2 subunit and hemerythrin have been solved, giving an accurate picture of the dinuclear iron centers in these proteins [20-221. The sMMO hydroxylase has recently been crystallized and its spacc group reported, but the structure has yet to be refined [23]. It is the ligands in the first coordination sphere of the iron atoms that most influence the redox potentials and other properties which determine the function of the active sites in each of these iron-oxo proteins. The first coordination sphere of R2 consists of mainly oxygen-donating ligands while that of hemerythrin has mainly N-donor ligands. In the absence of an hydroxylase crystal structure, overlaying the gene sequence of sMMO [24] on that of R2 has resulted in a plausible picture of the binuclear iron center of the former [25]. The proposed hydroxylase site resembles that of R2 much more t...

Fourier Transform Infrared Characterization of the Azido Complex of Methane Monooxygenase Hydroxylase from Methylococcus capsulatus (Bath)

“…Incubation of MMOH with a 50-fold excess of sodium azide results in the appearance of an ∼440-nm absorbance band in the UV−vis spectrum, which may be readily assigned to an azide-to-iron(III) charge-transfer band (Figure S1, Supporting Information) …”

“…2 Incubation of MMOH with a 50-fold excess of sodium azide results in the appearance of an ∼440-nm absorbance band in the UV-vis spectrum, which may be readily assigned to an azide-toiron(III) charge-transfer band (Figure S1, Supporting Information). 3 Attempts to define the geometry of azide binding by using X-ray crystallography were unsuccessful (Experimental Details, Supporting Information). Although a low-frequency band in the resonance Raman spectra of MMOH-N 3 was tentatively assigned to an Fe-N 3 stretching mode, the internal symmetric and asymmetric azido stretches could not be detected (Figure S2, Supporting Information).…”

THEORETICAL STUDY ON METHANE HYDROXYLATION BY MIMIC METHANE MONOOXYGENASE WITH bis(μ-OXO)DIMANGANESE CORE

“…In particular, manganese has been one of the most attractive candidates for replacing the native iron in these proteins, thanks to its rich redox chemistry that enables it to play a central role in a variety of biologically important metalloproteins. [15][16][17] In addition, it can exist in different oxidation states. Manganese(IV) is similar to iron(IV) in charge, ionic radius, and attractive magnetic property.…”

Characteristic features of the interaction between Fe(II) cations and the donor side of the manganese-depleted photosystem II

“…Apparently Fe cations are particularly bound to the high-affinity Mn-binding site. Indeed, the C-terminal sites of D1 and D2 proteins in the PSII reaction center (RC) comprise all the specific amino acid motifs [11] essential for binding and stabilizing both Fe and Mn cations in such enzymes as ribonucleotide reductase and methane monooxygenase [12,13]. Due to the blockade [14], at least two and no more than five Fe cations bound to the high-affinity Mn-binding site (and probably to other Mn-binding sites as well) make Y Z unavailable for the exogenous electron donors, such as Mn(II), DPC, and Fe(II).…”