Cytochrome P450 BM-3 (EC 1.14.14.1) catalyzes valuable oxygenation reactions for a broad range of industrially important substrates. Many of these substrates are poorly water-soluble, and P450 BM-3 is rapidly inactivated in presence of organic cosolvents. (Wong, T.S.; Arnold, F.H.; Schwaneberg, U. Biotechnol. Bioeng. 85 (3) 351-8.) Understanding how cosolvents reduce P450 BM-3 activity is of high academic and industrial interest. In a first attempt, we investigated the inactivation mechanism of DMSO by crystallizing P450 BM-3 heme domain (BMP) in 14% (v/v) and 28% (v/v) DMSO, denoted as Lo-DMSO and Hi-DMSO. The overall structures of Lo-DMSO (2.1 angstrom) and Hi-DMSO (1.7 angstrom) are similar to the reported structure (1BU7) in absence of DMSO. No indication of partial or global unfolding was found in the Lo-DMSO and Hi-DMSO structures as predicted by our previous molecular dynamics simulations and UV-vis measurements. In the Lo-DMSO structure, we observed a nonplanar distortion of heme and a displaced sixth water ligand. In the Hi-DMSO structure, the sixth water ligand was replaced by a DMSO molecule which is directly coordinated via its sulfur atom to the heme iron. Furthermore, the kink in the I-helix is more pronounced. The Lo- and Hi-DMSO structures suggest a cosolvent inactivation mechanism through covalent binding by the cosolvent DMSO to the heme iron.