Substrate-analogue-induced changes in the nickel-EPR spectrum of active methyl-coenzyme-M reductase from Methanobacterium thermoautotrophicum.
Methyl-coenzyme-M reductase (MCR) catalyzes the formation of methane from methyl-coenzyme M [2-(methylthio)ethanesulfonate] and 7-mercaptoheptanoylthreonine phosphate in methanogenic archaea. The enzyme contains the nickel porphinoid coenzyme F430 as a prosthetic group. In the active, reduced (red) state, the enzyme displays two characteristic EPR signals, MCR-red1 and MCR-red2, probably derived from Ni(I). In the presence of the substrate methyl-coenzyme M, the rhombic MCR-red2 signal is quantitatively converted to the axial MCR-red1 signal. We report here on the effects of inhibitory substrate analogues on the EPR spectrum of the enzyme. 3-Bromopropanesulfonate (BrPrSO3), which is the most potent inhibitor of MCR known to date (apparent Ki = 0.05 microM), converted the EPR signals MCR-red1 and MCR-red2 to a novel axial Ni(I) signal designated MCR-BrPrSO3. 3-Fluoropropanesulfonate (apparent Ki < 50 microM) and 3-iodopropanesulfonate (apparent Ki < 1 microM) induced a signal identical to that induced by BrPrSO3 without affecting the line shape, despite the fact that the fluorine, bromine and iodine isotopes employed have nuclear spins of I = 1/2, I = 3/2 and I = 5/2, respectively. This finding suggests that MCR-BrPrSO3 is not the result of a close halogen-Ni(I) interaction. 7-Bromoheptanoylthreonine phosphate (BrHpoThrP) (apparent Ki = 5 microM), which is an inhibitory substrate analogue of 7-mercaptoheptanoylthreonine phosphate, converted the signals MCR-red1 and MCR-red2 to a novel axial Ni(I) signal, MCR-BrHpoThrP, similar but not identical to MCR-BrPrSO3. The results indicate that inhibition of MCR by the halogenated substrate analogues investigated above is not via oxidation of Ni(I)F430. The different MCR EPR signals are assigned to different enzyme/substrate and enzyme/inhibitor complexes.
Rospert
S
S
Laboratorium für Mikrobiologie des Fachbereichs Biologie, Philipps-Universität, Marburg, Federal Republic of Germany.
Voges
M
M
Berkessel
A
A
Albracht
S P
SP
Thauer
R K
RK
D000476:Alkanesulfonates / Q000737:chemistry; D002384:Catalysis; D004578:Electron Spin Resonance Spectroscopy; D015080:Mesna / Q000031:analogs & derivatives / Q000378:metabolism; D008697:Methane / Q000378:metabolism; D017014:Methanobacterium / Q000201:enzymology*; D009532:Nickel / Q000737:chemistry*; D010088:Oxidoreductases / Q000302:isolation & purification / Q000378:metabolism*; D010769:Phosphothreonine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity
D000476:Alkanesulfonates; C049349:7-mercaptoheptanoylthreonine phosphate; D010769:Phosphothreonine; C078346:7-bromoheptanoylthreonine phosphate; C078347:3-fluoropropanesulfonate; C023349:methyl coenzyme M; D009532:Nickel; D010088:Oxidoreductases; C021572:methyl coenzyme M reductase; D015080:Mesna; D008697:Methane