Cyt P450 enzymes catalyze a diverse range of reactions including epoxidation, sulfoxidation, heteroatom dealkylation, C-C bond cleavage and C-C bond formation reactions as well as the oxidation of aliphatic and aromatic C-H bonds, a difficult reaction in environmental and biological chemistry. Site-directed mutagenesis combined with molecular spectroscopy on natural P450 monooxygenases provides with essential information delimiting the catalytic processes of P450s, Yet, possible undesired functional changes in H-bond networks related to substrate binding or intramolecular e- transfer, and/or allosteric effects could result from such point mutations. The catalytic activity of the heme iron in the active site of natural P450s is modulated and enhanced by the surrounding protein matrix, that is the 2nd coordination sphere (and beyond) and long-range H-bonding effects. Hence, we have chosen the advantageous use of de novo designed heme proteins, artificial scaffolds capturing the essential features of the heme active site of natural cyt P450 proteins, together with the required (surrounding) protein matrix (1). The resulting alpha-helical dimers of anti-parallel dimers with one thiolate heme per dimer has been used to explore the effects of 2nd coordination sphere. UV-Vis electronic absorption, EPR/HYSCORE and resonance Raman spectroscopies show that the scaffold bears a thiolate heme. Spectroscopic characterization of mutations in the de novo scaffolds tailored to modify the pKa of the Cys heme ligand, as well as the heme distal side and thiolate vs thiol ferrous heme, will be presented.