Mossbauer, EPR, and ENDOR Studies of the Hydroxylase and Reductase Components of Methane Monooxygenase from Methylosinus trichosporium OB3b

Soluble methane monooxygenase (MMO) isolated from Methylosinus trichosporium OB3b consists of three components: hydroxylase, reductase, and component B. The active-site diiron cluster of the hydroxylase has been studied with Mossbauer, ENDOR, and EPR spectroscopies. Mossbauer spectra of the oxidized cluster show that the two high-spin irons are antiferromagnetically coupled in accord with our preliminary study (Fox et al. J . Biol. Chem. 1988, 263, 10553-10556). Mossbauer studies also reveal the presence of two cluster conformations at pH 9. The excited-state S = 2 multiplet of the exchange-coupled cluster (Fe3+.Fe3+) gives rise to an integer-spin EPR signal near g = 8; this is the first quantitative study of such a signal from any system. Analysis of the temperature dependence of the g = 8 signal yields J = 15 f 5 cm-I for the exchange-coupling constant (Hex = JSleS2). This value is more than 1 order of magnitude smaller than those reported for the oxo-bridged clusters of hemerythrin and Escherichia coli ribonucleotide reductase (Hex = JSI.S2, J = 270 and 220 cm-I, respectively), suggesting that the bridging ligand of the hydroxylase cluster is not an unsubstituted oxygen atom. Mossbauer spectra of the hydroxylase in applied fields of up to 8 T reveal a paramagnetic admixture of a low-lying excited state into the ground singlet. Both the spectral shape and intensity are well represented by assuming that the spin expectation values for the cluster sites increase linearly with magnetic field. However, the origin of this effect is not fully explicable in the framework of the standard spin Hamiltonian including zero-field splittings and antisymmetric exchange. EPR studies of the uncomplexed mixed valence (Fe3+.Fe2+) hydroxylase show that it is composed of two slightly different cluster forms in an approximate 4: 1 ratio. The zero-field splitting (ZFS) of the ferrous site of the mixed valence hydroxylase is sensitive to complexation with products or inhibitors, while complexation by the component B perturbs the exchange coupling. The binding of the inhibitor dimethyl sulfoxide results in the smallest distribution of ZFS parameters and thus is investigated here in a correlated study using each of the three spectroscopic techniques. The data were analyzed with a spin Hamiltonian that includes exchange coupling ( J = 60 cm-l) and mixing of multiplets by zero-field splittings. The analysis shows that the orbital ground state of the ferrous site has predominantly d, symmetry; the z-axis of this orbital points along the z-direction of the cluster g-tensor. Mossbauer and 57Fe-ENDOR spectra indicate that the A-tensor of the ferric site is anisotropic; the 57Fe-ENDOR signals are the first reported for diiron-oxo clusters. Analysis of the Miissbauer spectra of the uncomplexed, reduced (Fe2+.Fe2+) hydroxylase cluster recorded in strong applied fields (up to 6.0 T) unambiguously shows that the two iron sites are inequivalent. Spectra of the oxidized cluster are also best fit by assuming that the irons of the cluster reside in inequivalent environments. Considered in light of the overall two-fold symmetry of hydroxylase revealed by ongoing structural studies, the present findings show that the hydroxylase contains two, probably identical, active-site diiron clusters whose individual iron atoms are structurally distinct. Mossbauer and EPR spectra of the [2Fe-2SI2+J+ cluster of the M M O reductase component are also reported and analyzed. Soluble methane monooxygenase (MMO, EC 1.14.13.25) consists of three protein components: a 40-kDa reductase containing both FAD and a [2Fe2S] cluster; a 16-kDa protein termed component B containing no metals or organic cofactors; and a 245-kDa hydroxylase (quaternary structure (afly)2) containing up to 4 mol of iron.] Soluble M M O catalyzes the 02-dependent oxidation of methane to methanoL2 In addition, a wide variety of other hydrocarbons are adventitiously ~ x i d i z e d . ~ Efficient reconstitution of NADH-linked catalytic turnover * Authors to whom correspondence should be directed. + Carnegie Mellon University. f University of Minnesota. (1) (a) Fox, B. G.; Froland, W. A.; Dege, J. E.; Lipscomb, J . D. J. Biol. Chem. 1989,264, 10023-10033. (b) Froland, W. F.; Andersson, K. K.; Lee, S.-K.; Liu, Y.; Lipscomb, J . D. In Applications of Enzyme Biotechnology; Kelly, J. W., Baldwin, T. O., Eds.; Plenum Press: New York, 1991; pp 39-53. (c) Fox, B. G.; Lipscomb, J. D. In Biological Oxidation Systems; Reddy, C . C . , Hamilton, G. A,, Madyastha, K. M., Eds.; Academic Press: New York, 1990; Vol. 1, pp 367-388. (2) Dalton, H. Adu. Appl. Microbiol. 1980, 26, 71-87. 0002-7863/93/1515-3688$04.00/0 requires all three protein component^.^ However, in the absence of the other components, the hydroxylase is able to catalyze hydroxylation reactions either upon chemical reduction and e x p o ~ u r e l ~ ~ ~ ~ to 0 2 or upon a d d i t i ~ n ~ ~ , ~ of H202, demonstrating that the complete active site required for oxygenase catalysis resides on the hydroxylase alone. All EPR and Mijssbauer studiesconducted to date are consistent with the presence of a spin-coupled diiron cluster in the hydroxylase active site.] Iron quantitation in combination with theobservation (3) (a) Rataj, M. J.; Knauth, J. E.; Donnelly, M. I. J. Biol. Chem. 1991, 266,18684-18690. (b) Fox, B.G.;Borneman, J.G.; Wackett,L.P.;Lipscomb, J. D. Biochemistry 1990, 29, 641945427. (c) Ruzicka, F.; Huang, D.-S.; Donnelly, M. I.; Frey, P. A. Biochemistry 1990, 29, 1696-1700. (d) Green, J.; Dalton, H. J. Biol. Chem. 1989, 246, 17698-17703. (4) (a) Fox, B. G.; Liu, Y.; Dege, J. E.; Lipscomb, J. D. J . Biol. Chem. 1991, 266, 540-550. (b) Froland, W. A.; Andersson, K. K.; Lee, S.-K.; Liu, Y.; Lipscomb, J. D. J . Biol. Chem. 1992, 267, 17588-17597. (c) Green, J.; Dalton, H. J. Biol. Chem. 1985, 260, 15795-15801, (5) Andersson, K. K.; Froland, W. A.; Lee, S.-K.; Lipscomb, J. D. New J. Chem. 1991, 15, 411-415.