Probing the reactivity of Ni in the active site of methyl-coenzyme M reductase with substrate analogues.
Methyl-coenzyme M reductase (MCR) catalyses the reduction of methyl-coenzyme M (CH(3)-S-CoM) with coenzyme B (HS-CoB) to methane and CoM-S-S-CoB. It contains the nickel porphyrinoid F(430) as prosthetic group which has to be in the Ni(I) oxidation state for the enzyme to be active. The active enzyme exhibits an axial Ni(I)-derived EPR signal MCR-red1. We report here on experiments with methyl-coenzyme M analogues showing how they affect the activity and the MCR-red1 signal of MCR from Methanothermobacter marburgensis. Ethyl-coenzyme M was the only methyl-coenzyme M analogue tested that was used by MCR as a substrate. Ethyl-coenzyme M was reduced to ethane (apparent K(M)=20 mM; apparent V(max)=0.1 U/mg) with a catalytic efficiency of less than 1% of that of methyl-coenzyme M reduction to methane (apparent K(M)=5 mM; apparent V(max)=30 U/mg). Propyl-coenzyme M (apparent K(i)=2 mM) and allyl-coenzyme M (apparent K(i)=0.1 mM) were reversible inhibitors. 2-Bromoethanesulfonate ([I](0.5 V)=2 micro M), cyano-coenzyme M ([I](0.5 V)=0.2 mM), 3-bromopropionate ([I](0.5 V)=3 mM), seleno-coenzyme M ([I](0.5 V)=6 mM) and trifluoromethyl-coenzyme M ([I](0.5 V)=6 mM) irreversibly inhibited the enzyme. In their presence the MRC-red1 signal was quenched, indicating the oxidation of Ni(I) to Ni(II). The rate of oxidation increased over 10-fold in the presence of coenzyme B, indicating that the Ni(I) reactivity was increased in the presence of coenzyme B. Enzyme inactivated in the presence of coenzyme B showed an isotropic signal characteristic of a radical that is spin coupled with one hydrogen nucleus. The coupling was also observed in D(2)O. The signal was abolished upon exposure of the enzyme to O(2). 3-Bromopropanesulfonate ([I](0.5 V)=0.1 micro M), 3-iodopropanesulfonate ([I](0.5 V)=1 micro M), and 4-bromobutyrate also inactivated MCR. In their presence the EPR signal of MCR-red1 was converted into a Ni-based EPR signal MCR-BPS that resembles in line shape the MCR-ox1 signal. The signal was quenched by O(2). 2-Bromoethanesulfonate and 3-bromopropanesulfonate, which both rapidly reacted with Ni(I) of MRC-red1, did not react with the Ni of MCR-ox1 and MCR-BPS. The Ni-based EPR spectra of both inactive forms were not affected in the presence of high concentrations of these two potent inhibitors.
Goenrich
Meike
M
Max-Planck-Institut für Terrestrische Mikrobiologie and Laboratorium für Mikrobiologie, Fachbereich Biologie, Philipps-Universität, Karl-von-Frisch-Strasse, 35043 Marburg, Germany.
Mahlert
Felix
F
Duin
Evert C
EC
Bauer
Carsten
C
Jaun
Bernhard
B
Thauer
Rudolf K
RK
D017738:Alkanesulfonic Acids / Q000378:metabolism; D001665:Binding Sites; D007700:Kinetics; D015080:Mesna / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D017013:Methanobacteriaceae / Q000201:enzymology; D008956:Models, Chemical; D015394:Molecular Structure; D009532:Nickel / Q000737:chemistry*; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D013379:Substrate Specificity
C495861:3-bromopropanesulfonate; D017738:Alkanesulfonic Acids; C018853:2-bromoethanesulfonic acid; D009532:Nickel; D010088:Oxidoreductases; C021572:methyl coenzyme M reductase; D015080:Mesna