pubs.acs.org/biochemistry Published on Web 12/30/2010 r 2010 American Chemical Society 1042 Biochemistry 2011, 50, 1042–1052 DOI: 10.1021/bi101559z Structural Design of the Active Site for Covalent Attachment of the Heme to the Protein Matrix: Studies on a Thermostable Cytochrome P450 † Sandeep Goyal, Megha S. Deshpande, and Shyamalava Mazumdar* Department of Chemical Sciences, Tata Institute of Fundamental Research, Homi Bhabha Road, Navy Nagar, Colaba, Mumbai 400 005, India Received September 25, 2010; Revised Manuscript Received December 24, 2010 ABSTRACT: The molecular basis of the post-translational modification involving covalent attachment of the heme with a glutamic acid observed in some enzymes of the CYP4 family of heme monooxygenases has been investigated using site-directed mutagenesis of CYP175A1 from Thermus thermophilus. Earlier studies of CYP4 as well as the G248E mutant of CYP101A1 showed covalent linkage of the heme to a conserved glutamic acid of helix I. We have introduced Glu/Asp at the Leu80 position in the β-turn of CYP175A1, on the basis of molecular modeling studies, to assess whether formation of such a covalent linkage is limited only to helix I or whether such modification may also take place with the residue that is spatially located at a position appropriate for activation by the heme peroxidase reaction. Tandem mass spectrometry analyses of the tryptic digest of the wild type and mutants of CYP175A1 were conducted to identify any heme-bound peptide. Tryptic digestion of the L80E mutant of CYP175A1 preincubated with H 2 O 2 showed formation of GLE(-heme)TDWGESWKEARK supporting covalent linkage of Glu80 with the heme in the mutant enzyme. No such heme-bound peptides were found if the sample was not preincubated in H 2 O 2 , indicating no activation of the Glu by the heme peroxidase reaction, as proposed earlier. The wild type or L80D mutant of the enzyme did not give any heme-bound peptide. Thus, the results support the idea that covalent attachment of the heme to an amino acid in the protein matrix depends on the structural design of the active site. The cytochrome P450 enzymes form a ubiquitous superfamily of heme b-containing monooxygenases, which are responsible for a large number of physiologically and biotechnologically im- portant transformations of numerous endogenous and exog- enous compounds (1-3). The crystal structure of most of the P450 enzymes shows that the active site of the enzyme consists of the heme 1 anchored to the protein matrix through a coordinate bond with a highly conserved cysteine (thiolate ligand) residue on its proximal site, which probably serves as the most important interaction between the heme and protein matrix. Also, heme binding in P450 enzymes is facilitated via other noncovalent interactions like ionic, hydrophobic, and van der Waals interac- tions between active site amino acid residues of the protein and functional groups of the porphyrin ring (2, 4). However, excep- tions are members of the CYP4 family of cytochrome P450 enzymes in which post-translational modification involving covalent attachment of the heme to the protein matrix has been observed in CYP4A, CYP4B, and CYP4F (not in CYP4F5 and -6) (1, 2, 5-8). In CYP4A3, the heme is attached to the protein via an ester link to Glu318, which is in helix I, and is predicted to be within the active site (9). The percentage of covalent linkage in CYP4A1, CYP4A3, CYP4A11, and CYP4B1 was found to decrease by more than 90% when the glutamic acid residue of helix I was mutated to alanine and other amino acid residues (1, 5). No covalent attachment, however, was found in CYP4F5, which has glycine at the respective position, but the G330E mutant of CYP4F5 shows the partial covalent attach- ment of the heme (6). In case of myeloperoxidase, two types of linkages are found, one involving an ester link between the Glu242 and Asp94 residues and the 1- and 5-methyl groups of heme, respectively, and the other a sulfonium link between the sulfur of Met243 and the β-carbon of the 2-vinyl group of heme (10, 11). Similarly, the lactoperoxidases are well-known to contain ester links of the heme 1- and 5-methyl groups with Glu and Asp residues (12-15). In eosinophil perox- idase, Glu241 and Asp93 have been shown to be involved in the formation of covalent links with the 1- and 5-methyl groups of heme, respectively (16). Not only the information described above but also numerous examples of the modifications of heme group are well-known and may or may not be involved in the same type of covalent linkage. Proteins that contains a covalent linkage between the heme prosthetic group and the protein matrix include cytochrome c, ascorbate peroxidase (APX), cytochrome c perox- idase (CcP), and thyroid peroxidase (11, 17-20). In cytochrome c, heme is covalently attached to the protein matrix through thioether bonds between two vinyl groups of the heme and the cysteine residues (17). In ascorbate peroxidase and cytochrome c peroxidase, covalent links between Trp41 and Trp51, respectively, and one of vinyl groups of heme were reported (18, 19). The presence of covalently bound heme in some enzymes of the CYP4 family and its absence in a vast majority of other P450 † Funding was provided by the Tata Institute of Fundamental Re- search and the Department of Biotechnology, Government of India. M. S.D. is a postdoctoral research fellow. *To whom correspondence should be addressed. E-mail: shyamal@ tifr.res.in. Telephone: 009122 22782363. Fax: 009122 2280 4610. 1 Abbreviations: heme, iron protoporphyrin IX regardless of oxida- tion and ligation state; WT, wild type; L80D and L80E, leucine 80 to aspartic acid and glutamic acid mutants of CYP175A1, respectively; CD, circular dichroism; T m , melting temperature; PDB, Research Collaboratory for Structural Bioinformatics (RCSB) Protein Data Bank.