Eubacterium acidaminophilum is able to conserve energy from reduction of glycine, betaine and sarcosine by synthesis of acetyl phosphate. This synthesis of acetyl phosphate is catalyzed by protein C of the reductase-complex. Release of acetyl phosphate by protein C takes place after reductive cleavage of carboxymethylselenoether bounded to selenoprotein A and formation of acetyl thioester at protein C. Number and position of reactive and sequenced thiols in the subunits GrdC and GrdD of protein C were still unclear. To investigate this, gene grdD1 of small subunit GrdD was cloned. 2 cysteine residues were analyzed at positions 98 and 359 in GrdD. Comparing gene products of grdC1, grdC2 and grdD1 with their homologues sequences in other glycine reducing organisms, GrdC has two conserved cysteines at positions 223 and 261 and GrdD one conserved cysteine at position 359. The homology of cysteine 261 in GrdC to catalytic cystein of beta-ketoacyl-acyl carrier protein synthase III was a first indication for an acetyl thioester formation at subunit GrdC followed by transacetylation to subunit GrdD. Analyzing enzyme activity by arsenate dependent hydrolysis of acetyl phosphate, GrdD as recombinant N-terminal Strep-tag-fusion showed enzyme activity, by contrast to C-terminal Strep-tag fusion of GrdC, that was completely inactive. Mutants GrdDC98→S and GrdDC359→A were expressed and analyzed in comparison to recombinant GrdD. Measuring enzyme activity by catalysis of arsenate-dependent hydrolysis of acetyl phosphate, GrdDC359→S was completely inactive, in contrast to GrdDC98→S, that was still active. After inactivation of GrdD by treatment with iodoacetate in presence or absence of substrate acetyl phosphate, thiols showed different modifications as was proved by peptide mapping and mass spectrometry, cystein 359 was carboxymethylated. After treatment of GrdD with substrate acetyl phosphate before iodo acetate GrdD was still full active. Cysteine 359 was unmodified, therefore it could be labeled by 4 vinyl-pyridine. Cysteine 98 was not accessible to carboxymethylation by iodo acetate in the native enzyme in the presence or absence of the substrate, but could be alkylated after denaturation. This clearly confirmed the catalytic role of cysteine 359 as the active site thiol of GrdD responsible for release of acetyl phosphate. By atom absorption spectrometry zinc, was found in the recombinant subunit GrdC. The possible function of zinc as cofactor of GrdC in catalyzing a transfer of carboxymethyl group from selenoprotein A to GrdD was discussed.