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The Corrinoid‐Containing 23‐kDa Subunit MtrA of the Energy‐Conserving N 5 ‐Methyltetrahydromethanopterin:coenzyme M Methyltransferase Complex from Methanobacterium Thermoautotrophicum

1996; Wiley; Volume: 241; Issue: 1 Linguagem: Inglês

10.1111/j.1432-1033.1996.0149t.x

1432-1033

Ulrike Harms, Rudolf K. Thauer,

Metalloenzymes and iron-sulfur proteins

N 5 ‐Methyltetrahydromethanopterin:coenzyme M methyltransferase (Mtr) from Methanobacterium thermoautotrophicum is a membrane‐associated enzyme complex that catalyzes an energy‐conserving, sodium ion translocating step in methanogenesis from H 2 and CO 2 . The complex is composed of eight different subunits, MtrA–H 1 one of which (MtrA) harbours a corrinoid as prosthetic group. In this study, we report the structural properties of MtrA1[des‐(214–239)‐MtrA], which is a deletion mutant of MtrA that lacks the last 25 C‐terminal hydrophobic amino acids rendering the membrane protein soluble: (a) mtrAl was heterologously expressed in Escherichia coli. Overexpression yielded a cytoplasmic protein which was purified approximately tenfold to apparent homogeneity. The purified protein was devoid of its corrinoid prosthetic group and not correctly folded as was evident from its electrophoretic mobility in SDS/PAGE. (b) Unfolding of MtrA1 with guanidine/HCl and refolding in the presence of cobalamin resulted in the formation of the correctly folded MtrA1 holoprotein that contained tightly bound cob(II)‐alamin; the rate of reconstitution was highest when the refolding proceeded in the presence of titanium(III) citrate, which suggested that cob(1)alamin is the corrinoid species that binds to the apoprotein. (c) EPR spectra of the cob(II)alamin‐containing holoprotein differentially labelled with 14 N (nuclear spin 1) and 15 N (nuclear spin 1/2) revealed that the corrinoid is bound to MtrA1 in the base‐off form and that the Co(II) of the prosthetic group is coordinated by a histidine residue of the apoprotein. The results are interpreted with respect to the mechanism of energy conservation by the MtrA–H complex.