With these highly reproducible data in a biological matrix in hand, the new MISER-GC-MS technique was applied to YfeX in a whole cell screening of a focused mutant library. The dye-decolorizing peroxidase YfeX from E. coli was previously shown to perform non-natural carbene-transfer reactions such as carbonyl olefination and C-H functionalisation. The starting activity for the latter reaction was previously improved by an alanine scan within the active site. We were subsequently interested in the influence of the axial ligand in the activity of YfeX on the C-H functionalisation reaction. The axial ligand complexes the heme iron and substantially influences its redox potential and electrophilicity and hence the overall activity of the occurring heme-carbenoid complex. Amino acids having side chains, which are similar in size to wildtype histidine and harbour a lone electron pair-i.e. serine, threonine, cysteine-are preferred and others will most likely result in complete loss of activity. To study the axial ligand in YfeX, we saturated the axial ligand residue histidine 215-using Golden Mutagenesis -and screened the resulting library for the occurrence of other functional axial ligands in whole cell reactions by stacked MISER-GC-MS using the carbene-transfer reaction on 1-methyl-indole. As a control for the background reaction cells harbouring the empty plasmid (pAGM22082) were included in this plate in column 6. In column 7 the parental YfeX variant was placed as positive control exhibiting H215 as natural axial heme ligand. To our delight, the results demonstrated the feasibility of MISER-GC-MS for the screening of this reaction. The parental YfeX variant could be clearly distinguished from the background reaction included as empty plasmid control (Figure ). A new variant was identified, which carried a highly unusual tryptophan as axial heme ligand. To proof that the MISERgram revealed a "true positive" result, the corresponding plasmid was freshly transformed, expressed and purified by His-Tag chromatography. These results confirmed YfeX-H215W showing only slightly reduced activities compared to the parental variant (Figure ).