Abstract
The soluble form of methane monooxygenase (MMO) consists of three components: reductase, hydroxylase (MMOH), and "B" (MMOB). Resting MMOH contains a diferric bis-mu-hydroxodinuclear iron "diamond core" cluster which is the site of oxygen activation chemistry after reduction. Here it is shown that gamma-irradiation of MMOH at 77 K results in reduction of the diiron cluster to an EPR active Fe(II). Fe(III) mixed valence state. At this temperature, the conformation of the enzyme remains essentially unchanged during reduction, so the EPR-spectrum becomes a probe of the conformation of the diferric state. The gamma-irradiated MMOH exhibits EPR spectra that differ in lineshape and saturation properties from those of the mixed valence MMOH generated by chemical reduction in solution; annealing the gamma-irradiated sample at 230 K yields the spectra of the chemically reduced sample. This demonstrates that the conformation of diferric MMOH in the vicinity of the diiron cluster changes during reduction to the mixed valence state. The analogous experiment for the MMOB.MMOH complex gives a new mixed valence species following gamma-irradiation that differs from all previously reported mixed valence species. Thus, binding of MMOB also causes a change in the conformation of diferric MMOH. It is hypothesized that the structural changes observed for the first time here may involve conversion of the dihydroxo-bridged diamond core structure to one with more readily dissociable bridging oxygen ligands to facilitate reaction with O2 following cluster reduction.
Highlights
EXPERIMENTAL PROCEDURESMMOH and MMOB from Methylosinus trichosporium OB3b were prepared as previously reported [11], and the specific activities were over 600 and 9000 milliunits/mg of protein for furan oxidation at room temperature, respectively
The soluble form of methane monooxygenase (MMO) consists of three components: reductase, hydroxylase (MMOH), and “B” (MMOB)
The soluble form of MMO is composed of three different protein components, each of which is required for efficient catalysis in vivo: a monomeric 38-kDa reductase (MMOR) with iron-sulfur (Fe2S2) and FAD cofactors, a 248-kDa hydroxylase (MMOH) containing bis--hydroxo-bridged “diamond core” dinuclear iron clusters in an (␣␥)2 quaternary structure [7,8,9,10], and a monomeric 15-kDa “B” component (MMOB) without cofactors or metals
Summary
MMOH and MMOB from Methylosinus trichosporium OB3b were prepared as previously reported [11], and the specific activities were over 600 and 9000 milliunits/mg of protein for furan oxidation at room temperature, respectively. The chemical reduction of MMOH to the mixed valence state was achieved by incubating the oxidized protein with 1.1 reducing equivalents of buffered sodium dithionite and phenazine methosulfate under anaerobic conditions as described elsewhere [11, 15]. The ␥-irradiation of the samples was performed in quartz tubes with internal diameters of about 3 mm immersed in liquid nitrogen in a Dewar that was exposed to ␥-rays in a 137Cs source giving a dose rate of 65 kilorads/h. Quantitations of the mixed valent EPR signals were done as described previously under nonsaturating microwave power conditions [21] and by the methods described by Aasa and Vanngård [23]. The irradiation induced strong radical signals that distorted both the base line and the g1 values of the mixed valent iron cluster signals, making the quantitations less accurate than expected for typical EPR samples. The data were analyzed using the equation, log (I/ߛP) ϭ a Ϫ (b/2)(log (P1⁄2 Ϫ log P), from which the half saturation power P1⁄2 was determined graphically [24]
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