Using a multiple alignment of 175 cytochrome P450 (CYP) family 2 sequences, 20 conserved sequence motifs (CSMs) were identified with the program PCPMer. Functional importance of the CSM in CYP2B enzymes was assessed from available data on site-directed mutants and genetic variants. These analyses suggested an important role of the CSM 8, which corresponds to(187)RFDYKD(192) in CYP2B4. Further analysis showed that residues 187, 188, 190, and 192 have a very high rank order of conservation compared with 189 and 191. Therefore, eight mutants (R187A, R187K, F188A, D189A, Y190A, K191A, D192A, and a negative control K186A) were made in an N-terminal truncated and modified form of CYP2B4 with an internal mutation, which is termed 2B4dH/H226Y. Function was examined with the substrates 7-methoxy-4-(trifluoromethyl)coumarin (7-MFC), 7-ethoxy-4-(trifluoromethyl)coumarin (7-EFC), 7-benzyloxy-4-(trifluoromethyl)coumarin (7-BFC), and testosterone and with the inhibitors 4-(4-chlorophenyl)imidazole (4-CPI) and bifonazole (BIF). Compared with the template and K186A, the mutants R187A, R187K, F188A, Y190A, and D192A showed > or =2-fold altered substrate specificity, k(cat), K(m), and/or k(cat)/K(m) for 7-MFC and 7-EFC and 3- to 6-fold decreases in differential inhibition (IC(50,BIF)/IC(50,4-CPI)). Subsequently, these mutants displayed 5-12 degrees C decreases in thermal stability (T(m)) and 2-8 degrees C decreases in catalytic tolerance to temperature (T(50)) compared with the template and K186A. Furthermore, when R187A and D192A were introduced in CYP2B1dH, the P450 expression and thermal stability were decreased. In addition, R187A showed increased activity with 7-EFC and decreased IC(50,BIF)/IC(50,4-CPI) compared with 2B1dH. Analysis of long range residue-residue interactions in the CYP2B4 crystal structures indicated strong hydrogen bonds involving Glu(149)-Asn(177)-Arg(187)-Tyr(190) and Asp(192)-Val(194), which were significantly-reduced/abolished by the Arg(187)-->Ala and Asp(192)-->Alasubstitutions, respectively.