Proline residues in transmembrane alpha helices affect the folding of bacteriorhodopsin

Proline residues occur frequently in transmembrane alpha helices, which contrasts with their behaviour as helix-breakers in water-soluble proteins. The three membrane-embedded proline residues of bacteriorhodopsin have been replaced individually by alanine and glycine to give P50A, or P50G on helix B, P91A, or P91G on helix C, and P186A or P186G on helix F, and the effect on the protein folding kinetics has been investigated. The rate-limiting apoprotein folding step, which results in formation of a seven transmembrane, alpha helical state, was slower than wild-type protein for the Pro50 and Pro91 mutants, regardless of whether they were mutated to Ala or Gly. These proline residues give rise to several inter-helix contacts, which are therefore important in folding to the seven transmembrane helix state. No evidence for cis-trans isomerisations of the peptidyl prolyl bonds was found during this rate-limiting apoprotein folding step. Mutations of all three membrane-embedded proline residues affected the subsequent retinal binding and final folding to bacteriorhodopsin, suggesting that these proline residues contribute to formation of the retinal binding pocket within the helix bundle, again via helix/helix interactions. These results point to proline residues in transmembrane alpha helices being important in the folding of integral membrane proteins. The helix/helix interactions and hydrogen bonds that arise from the presence of proline residues in transmembrane alpha helices can affect the formation of transmembrane alpha helix bundles as well as cofactor binding pockets.     

Atomic structure of Mycobacterium tuberculosis CYP121 to 1.06 A reveals novel features of cytochrome P450

The first structure of a P450 to an atomic resolution of 1.06 A has been solved for CYP121 from Mycobacterium tuberculosis. A comparison with P450 EryF (CYP107A1) reveals a remarkable overall similarity in fold with major differences residing in active site structural elements. The high resolution obtained allows visualization of several unusual aspects. The heme cofactor is bound in two distinct conformations while being notably kinked in one pyrrole group due to close interaction with the proline residue (Pro(346)) immediately following the heme iron-ligating cysteine (Cys(345)). The active site is remarkably rigid in comparison with the remainder of the structure, notwithstanding the large cavity volume of 1350 A(3). The region immediately surrounding the distal water ligand is remarkable in several aspects. Unlike other bacterial P450s, the I helix shows no deformation, similar to mammalian CYP2C5. In addition, the positively charged Arg(386) is located immediately above the heme plane, dominating the local structure. Putative proton relay pathways from protein surface to heme (converging at Ser(279)) are identified. Most interestingly, the electron density indicates weak binding of a dioxygen molecule to the P450. This structure provides a basis for rational design of putative antimycobacterial agents.     

Molecular evolution of the insect Halloween family of cytochrome P450s: phylogeny, gene organization and functional conservation

The insect molting hormone, 20-hydroxyecdysone (20E), is a major modulator of the developmental processes resulting in molting and metamorphosis. During evolution selective forces have preserved the Halloween genes encoding cytochrome P450 (P450) enzymes that mediate the biosynthesis of 20E. In the present study, we examine the phylogenetic relationships of these P450 genes in holometabolous insects belonging to the orders Hymenoptera, Coleoptera, Lepidoptera and Diptera. The analyzed insect genomes each contains single orthologs of Phantom (CYP306A1), Disembodied (CYP302A1), Shadow (CYP315A1) and Shade (CYP314A1), the terminal hydroxylases. In Drosophila melanogaster, the Halloween gene spook (Cyp307a1) is required for the biosynthesis of 20E, although a function has not yet been identified. Unlike the other Halloween genes, the ancestor of this gene evolved into three paralogs, all in the CYP307 family, through gene duplication. The genomic stability of these paralogs varies among species. Intron-exon structures indicate that D. melanogaster Cyp307a1 is a mRNA-derived paralog of spookier (Cyp307a2), which is the ancestral gene and the closest ortholog of the coleopteran, lepidopteran and mosquito CYP307A subfamily genes. Evolutionary links between the insect Halloween genes and vertebrate steroidogenic P450s suggest that they originated from common ancestors, perhaps destined for steroidogenesis, before the deuterostome-arthropod split. Conservation of putative substrate recognition sites of orthologous Halloween genes indicates selective constraint on these residues to prevent functional divergence. The results suggest that duplications of ancestral P450 genes that acquired novel functions may have been an important mechanism for evolving the ecdysteroidogenic pathway.     

Characterization of active site structure in CYP121. A cytochrome P450 essential for viability of Mycobacterium tuberculosis H37Rv

Mycobacterium tuberculosis (Mtb) cytochrome P450 gene CYP121 is shown to be essential for viability of the bacterium in vitro by gene knock-out with complementation. Production of CYP121 protein in Mtb cells is demonstrated. Minimum inhibitory concentration values for azole drugs against Mtb H37Rv were determined, the rank order of which correlated well with Kd values for their binding to CYP121. Solution-state spectroscopic, kinetic, and thermodynamic studies and crystal structure determination for a series of CYP121 active site mutants provide further insights into structure and biophysical features of the enzyme. Pro346 was shown to control heme cofactor conformation, whereas Arg386 is a critical determinant of heme potential, with an unprecedented 280-mV increase in heme iron redox potential in a R386L mutant. A homologous Mtb redox partner system was reconstituted and transported electrons faster to CYP121 R386L than to wild type CYP121. Heme potential was not perturbed in a F338H mutant, suggesting that a proposed P450 superfamily-wide role for the phylogenetically conserved phenylalanine in heme thermodynamic regulation is unlikely. Collectively, data point to an important cellular role for CYP121 and highlight its potential as a novel Mtb drug target.     

Comparing the electronic properties and docking calculations of heme derivatives on CYP2B4

Cytochrome P-450 is a group of enzymes involved in the biotransformation of many substances, including drugs. These enzymes possess a heme group (1) that when it is properly modified induces several important physicochemical changes that affect their enzymatic activity. In this work, the five structurally modified heme derivatives 2–6 and the native heme 1 were docked on CYP2B4, (an isoform of P450), in order to determine whether such modifications alter their binding form and binding affinity for CYP2B4 apoprotein. In addition, docking calculations were used to evaluate the affinity of CYP2B4 apoprotein-heme complexes for aniline (A) and N-methyl-aniline (NMA). Results showing the CYP2B4 heme 4- and heme 6-apoprotein complexes to be most energetically stable indicate that either hindrance effects or electronic properties are the most important factors with respect to the binding of heme derivatives at the heme-binding site. Furthermore, although all heme-apoprotein complexes demonstrated high affinity for both A and NMA, the CYP2B4 apoprotein-5 complex had higher affinity for A, and the heme 6 complex had higher affinity for NMA. Finally, surface electronic properties (SEP) were calculated in order to explain why certain arginine residues of CYP2B4 apoprotein interact with polarizable functionalities, such as ester groups or sp ² carbons, present in some heme derivates. The main physicochemical parameter involved in the recognition process of the heme derivatives, the CYP2B4 apoprotein and A or NMA, are reported. Figure Scheme of steps to be followed for obtaining five new CYP2B4 apoprotein-heme complexes by docking     

Probing the structure of the linker connecting the reductase and heme domains of cytochrome P450BM-3 using site-directed mutagenesis.

Cytochrome P450BM-3 is a catalytically self-sufficient fatty acid hydroxylase containing one equivalent each of heme, FMN, and FAD. The heme and flavins reside in separate domains connected by a linker peptide. In an earlier study (Govindaraj S, Poulos T, 1995, Biochemistry 34:11221-11226), we found that the length but not the sequence of the linker connecting the heme and reductase domains is important for enzyme activity. In the present study, residues in the linker were replaced with Pro and Gly to probe the role that regular secondary structure plays in linker function. The rate of flavin-to-heme electron transfer and the fatty acid hydroxylase activities of the glycine and proline substitution mutants, including a six-proline substitution, did not change significantly relative to wild-type enzyme. These results indicate that the linker does not adopt any regular secondary structure essential for activity and that the length of the linker is the critical feature that controls flavin-to-heme electron transfer.     

Site-directed mutagenesis of the putative distal helix of peroxygenase cytochrome P450

CYP152A1 is an unusual, peroxygenase enzyme that catalyzes the beta- or alpha-hydroxylation of fatty acids by efficiently introducing an oxygen atom from H2O2 to the fatty acid. To clarify the mechanistic roles of amino acid residues in this enzyme, we have used site-directed mutagenesis of residues in the putative distal helix and measured the spectroscopic and enzymatic properties of the mutant proteins. Initially, we carried out Lys-scanning mutagenesis of amino acids in this region to determine residues of CYP152A1 that might have a mechanistic role. Among the Lys mutants, only P243K gave an absorption spectrum characteristic of a nitrogenous ligand-bound form of a ferric P450. Further investigation of the Pro243 site revealed that a P243H mutant also exhibited a nitrogen-bound form, but that the mutants P243A or P243S did not. On the hydroxylation of myristic acid by the Lys mutants, we observed a large decrease in activity for R242K and A246K. We therefore examined other mutants at amino acid positions 242 and 246. At position 246, an A246K mutant showed a roughly 19-fold lower affinity for myristic acid than the wild type. Replacing Ala246 with Ser decreased the catalytic activity, but did not affect affinity for the substrate. An A246V mutant showed slightly reduced activity and moderately reduced affinity. At position 242, an R242A showed about a fivefold lower affinity than the wild type for myristic acid. The Km values for H2O2 increased and Vmax values decreased in the order of wild type, R242K, and R242A when H2O2 was used; furthermore, Vmax/Km was greatly reduced in R242A compared with the wild type. If cumene hydroperoxide was used instead of H2O2, however, the Km values were not affected much by these substitutions. Together, our results suggest that in CYP152A1 the side chain of Pro243 is located close to the iron at the distal side of a heme molecule; the fatty acid substrate may be positioned near to Ala246 in the catalytic pocket, although Ala246 does not participate in hydrophobic interactions with the substrate; and that Arg242 is a critical residue for substrate binding and H2O2-specific catalysis.     

Structure-function relationships of human aromatase cytochrome P-450 using molecular modeling and site-directed mutagenesis

The conversion of androgens to estrogens is catalyzed by an enzyme complex named aromatase, which consists of a form of cytochrome P-450, aromatase cytochrome P-450 (cytochrome P-450AROM), and the flavoprotein, NADPH-cytochrome P-450 reductase. As a first step toward investigation of the structure-function relationships of cytochrome P-450AROM, we have used computer modeling to align the amino acid sequence of cytochrome P-450AROM with that of cytochrome P-450CAM from Pseudomonas putida and thus create a substrate pocket using the heme-binding region and the I-helix of cytochrome P-450CAM as the template. Site-directed mutagenesis was then carried out at two sites: one at a region that aligns with the bend in the I-helix of cytochrome P-450CAM and the other at a glutamate (Glu302) just N-terminal of this bend, which is predicted to be in close proximity to the C2-position of the androstenedione substrate. To determine the importance of the former region, three mutants were constructed: A307G (Ala307----Gly), P308V (Pro308----Val), and GAGV, which changed -Ile305-Ala306-Ala307-Pro308- to -Gly-Ala-Gly-Val- (the corresponding sequence found in 17 alpha-hydroxylase cytochrome P-450). When these proteins were expressed in COS-1 cells, it was found that the activity of P308V was approximately one-third that of the wild type. These observations are consistent with the concept that Pro308 causes a bend in the I-helix of cytochrome P-450AROM, similar to that observed in cytochrome P-450CAM, which is believed to be important in forming the substrate-binding pocket. The next set of mutants were designed to determine the importance of Glu302 in catalysis. Four mutants were prepared in which Glu302 was changed either to Ala, Val, Gln, or Asp, and the activities of the expressed proteins were examined. It was found that mutations in which the carboxylic acid was replaced were essentially devoid of activity. On the other hand, changing Glu302 to Asp resulted in a two-thirds reduction in the apparent Vmax. These results support the role of a carboxylic acid residue at position 302 in the catalytic activity of cytochrome P-450AROM.     

Covalent linkage of prosthetic heme to CYP4 family P450 enzymes.

An extensive body of research on the structural properties of cytochrome P450 enzymes has established that these proteins possess a b-type heme prosthetic group which is noncovalently bound at the active site. Coordinate, electrostatic, and hydrogen bond interactions between the protein backbone and heme functional groups are readily overcome upon mild acid treatment of the enzyme, which releases free heme from the protein. In the present study, we have used a combination of HPLC, LC/ESI-MS, and SDS-PAGE techniques to demonstrate that members of the mammalian CYP4B, CYP4F, and CYP4A subfamilies bind their heme in an unusually tight manner. HPLC chromatography of CYP4B1 on a POROS R2 column under mild acidic conditions caused dissociation of less than one-third of the heme from the protein. Moreover, heme was not substantially removed from CYP4B1 under electrospray or electrophoresis conditions that readily release the prosthetic group from other non-CYP4 P450 isoforms. This was evidenced by an intact protein mass value of 59,217 +/- 3 amu for CYP4B1 (i.e., apoprotein plus heme) and extensive staining of this approximately 60 kDa protein with tetramethylbenzidine/H(2)O(2) following SDS-PAGE. In addition, treatment of CYP4B1, CYP4F3, and CYP4A5/7 with strong base generated a new, chromatographically distinct, polar heme species with a mass of 632.3 amu rather than 616.2 amu. This mass shift is indicative of the incorporation of an oxygen atom into the heme nucleus and is consistent with the presence of a novel covalent ester linkage between the protein backbone of the CYP4 family of mammalian P450s and their heme catalytic center.     

Crystal structure of the Mycobacterium tuberculosis P450 CYP121-fluconazole complex reveals new azole drug-P450 binding mode

Azole and triazole drugs are cytochrome P450 inhibitors widely used as fungal antibiotics and possessing potent antimycobacterial activity. We present here the crystal structure of Mycobacterium tuberculosis cytochrome P450 CYP121 in complex with the triazole drug fluconazole, revealing a new azole heme ligation mode. In contrast to other structurally characterized cytochrome P450 azole complexes, where the azole nitrogen directly coordinates the heme iron, in CYP121 fluconazole does not displace the aqua sixth heme ligand but occupies a position that allows formation of a direct hydrogen bond to the aqua sixth heme ligand. Direct ligation of fluconazole to the heme iron is observed in a minority of CYP121 molecules, albeit with severe deviations from ideal geometry due to close contacts with active site residues. Analysis of both ligand-on and -off structures reveals the relative position of active site residues derived from the I-helix is a key determinant in the relative ratio of on and off states. Regardless, both ligand-bound states lead to P450 inactivation by active site occlusion. This previously unrecognized means of P450 inactivation is consistent with spectroscopic analyses in both solution and in the crystalline form and raises important questions relating to interaction of azoles with both pathogen and human P450s.     

Importance of a proline-rich sequence in the amino-terminal region for correct folding of mitochondrial and soluble microbial p450s

All microsomal P450s have a proline-rich sequence (PR) in the amino-terminal region that is needed for proper folding [Kusano, K., Sakaguchi, M., Kagawa, N., Waterman, M.R. and Omura, T. (2001) J. Biochem., 129, 259-269]. There are also multiple proline residues near the amino-termini of the mature forms of all mitochondrial P450s and the amino-termini of soluble microbial P450s. To examine the functional significance of the PR in mitochondrial P450s, we expressed human P450c27 (CYP27) and bovine P450scc (CYP11A1) in an Escherichia coli heterologous expression system, and found that in each one specific proline residue is important for correct folding. Deletions from the amino-terminus further indicated the importance of the PR for the expression of a spectrally normal P450c27. Essentially the same results were obtained with two soluble microbial P450s, P450cam (CYP101) and P450nor, in each of which a PR is important for proper folding. We conclude that in all P450s (mitochondrial, microbial and microsomal P450s), a proline-rich sequence located in the amino-terminal region is important for proper folding. Furthermore, we predict that the importance of the PR in P450 folding is to reduce the tendency of the polypeptide to misfold prior to heme binding.     

Analysis of conserved glutamate residues in Porphyromonas gingivalis outer membrane receptor HmuR: toward a further understanding of heme uptake

The aim of this study was to broaden the current knowledge about the Porphyromonas gingivalis heme receptor HmuR. Site-directed mutagenesis was employed to replace Glu427, Glu448, Glu458 and Glu503 by alanines and to construct a triple Glu427Ala/Glu448Ala/Glu 458Ala mutant. All iron/heme-starved P. gingivalis mutants showed decreased growth recovery when human serum as the iron/heme source was used, hmuR::ermF, hmuR ^E503A and hmuR ^{E427A,E448A,E458A} mutant strains being the most affected. E. coli cells expressing HmuR with mutated glutamate residues bound hemin, hemoglobin and hemin–serum albumin complex with the same efficiency as did the wild-type recombinant protein, suggesting that the residues were not directly involved in heme binding. These data indicate that in addition to two conserved histidine residues (His95 and His434), NPDL and YRAP motifs, conserved glutamate residues are important for HmuR to utilize heme present in serum hemoproteins.     

Protein recognition in ferredoxin–P450 electron transfer in the class I CYP199A2 system from Rhodopseudomonas palustris

CYP199A2 from Rhodopseudomonas palustris CGA009 is a heme monooxygenase that catalyzes the oxidation of para-substituted benzoic acids. CYP199A2 activity is reconstituted by a class I electron transfer chain consisting of the associated [2Fe–2S] ferredoxin palustrisredoxin (Pux) and a flavoprotein palustrisredoxin reductase (PuR). Another [2Fe–2S] ferredoxin, palustrisredoxin B (PuxB; RPA3956) has been identified in the genome. PuxB shares sequence identity and motifs with vertebrate-type ferredoxins involved in Fe–S cluster assembly but also 50% identity with Pux and it mediates electron transfer from PuR to CYP199A2, albeit with lower steady-state turnover activity: 99 nmol (nmol P450)^?1min^?1 for 4-methoxybenzoic acid oxidation compared with 1,438 nmol (nmol P450)^?1 min^?1 for Pux. This difference mainly arises from weak CYP199A2–PuxB binding (K _m 34.3 vs. 0.45 μM for Pux) rather than slow electron transfer (k _cat 19.1 vs. 37.9 s^?1 for Pux). Comparison of the 2.0-Å-resolution crystal structure of the PuxB A105R mutant with other vertebrate-type, P450-associated ferredoxins revealed similar protein folds but also significant differences in some loop regions. Therefore, PuxB offers a platform for studying ferredoxin–P450 recognition in class I P450 systems. Substitution of PuxB residues at key locations with those in Pux shows that Ala42, Cys43, and Ala44 in the [2Fe–2S] cluster binding loop and Met66 are important in electron transfer from PuxB to CYP199A2, whereas Phe73 and the C-terminal Ala105 were involved in both protein binding and electron transfer.     

Rapid P450 heme iron reduction by laser photoexcitation of Mycobacterium tuberculosis CYP121 and CYP51B1. Analysis of CO complexation reactions and reversibility of the P450/P420 equilibrium

We demonstrate that photoexcitation of NAD(P)H reduces heme iron of Mycobacterium tuberculosis P450s CYP121 and CYP51B1 on the microsecond time scale. Rates of formation for the ferrous-carbonmonoxy (Fe(II)-CO) complex were determined across a range of coenzyme/CO concentrations. CYP121 reaction transients were biphasic. A hyperbolic dependence on CO concentration was observed, consistent with the presence of a CO binding site in ferric CYP121. CYP51B1 absorption transients for Fe(II)-CO complex formation were monophasic. The reaction rate was second order with respect to [CO], suggesting the absence of a CO-binding site in ferric CYP51B1. In the absence of CO, heme iron reduction by photoexcited NAD(P)H is fast ( approximately 10,000-11,000 s(-1)) with both P450s. For CYP121, transients revealed initial production of the thiolate-coordinated (P450) complex (absorbance maximum at 448 nm), followed by a slower phase reporting partial conversion to the thiol-coordinated P420 species (at 420 nm). The slow phase amplitude increased at lower pH values, consistent with heme cysteinate protonation underlying the transition. Thus, CO binding occurs to the thiolate-coordinated ferrous form prior to cysteinate protonation. For CYP51B1, slow conversions of both the ferrous/Fe(II)-CO forms to species with spectral maxima at 423/421.5 nm occurred following photoexcitation in the absence/presence of CO. This reflected conversion from ferrous thiolate- to thiol-coordinated forms in both cases, indicating instability of the thiolate-coordinated ferrous CYP51B1. CYP121 Fe(II)-CO complex pH titrations revealed reversible spectral transitions between P450 and P420 forms. Our data provide strong evidence for P420 formation linked to reversible heme thiolate protonation, and demonstrate key differences in heme chemistry and CO binding for CYP121 and CYP51B1.     

Crystal Structure of CYP24A1, a Mitochondrial Cytochrome P450 Involved in Vitamin D Metabolism

Cytochrome P450 (CYP) 24A1 catalyzes the side-chain oxidation of the hormonal form of vitamin D. Expression of CYP24A1 is up-regulated to attenuate vitamin D signaling associated with calcium homeostasis and cellular growth processes. The development of therapeutics for disorders linked to vitamin D insufficiency would be greatly facilitated by structural knowledge of CYP24A1. Here, we report the crystal structure of rat CYP24A1 at 2.5 Å resolution. The structure exhibits an open cleft leading to the active-site heme prosthetic group on the distal surface that is likely to define the path of substrate access into the active site. The entrance to the cleft is flanked by conserved hydrophobic residues on helices A′ and G′, suggesting a mode of insertion into the inner mitochondrial membrane. A docking model for 1α,25-dihydroxyvitamin D₃ binding in the open form of CYP24A1 that clarifies the structural determinants of secosteroid recognition and validates the predictive power of existing homology models of CYP24A1 is proposed. Analysis of CYP24A1's proximal surface identifies the determinants of adrenodoxin recognition as a constellation of conserved residues from helices K, K″, and L that converge with an adjacent lysine-rich loop for binding the redox protein. Overall, the CYP24A1 structure provides the first template for understanding membrane insertion, substrate binding, and redox partner interaction in mitochondrial P450s.     

Folding requirements are different between sterol 14alpha-demethylase (CYP51) from Mycobacterium tuberculosis and human or fungal orthologs

Upon sequence alignment of CYP51 sterol 14alpha-demethylase from animals, plants, fungi, and bacteria, arginine corresponding to Arg-448 of CYP51 in Mycobacterium tuberculosis (MT) is conserved near the C terminus of all family members. In MTCYP51 Arg-448 forms a salt bridge with Asp-287, connecting beta-strand 3-2 with helix J. Deletion of the three C-terminal residues of MTCYP51 has little effect on expression of P450 in Escherichia coli. However, truncation of the fourth amino acid (Arg-448) completely abolishes P450 expression. We have investigated whether Arg-448 has other structural or functional roles in addition to folding and whether its conservation reflects conservation of a common folding pathway in the CYP51 family. Characterization of wild type protein and three mutants, R448K, R448I, and R448A, including examination of catalytic activity, secondary and tertiary structure analysis by circular dichroism and tryptophan fluorescence, and studies of both equilibrium and temporal MTCYP51 unfolding behavior, shows that Arg-448 does not play any role in P450 function or maintenance of the native structure. C-terminal truncation of Candida albicans and human CYP51 orthologs reveals that, despite conservation in sequence, the requirement for arginine at the homologous C-terminal position in folding in E. coli is not conserved. Thus, despite similar spatial folds, functionally related but evolutionarily distinct P450s can follow different folding pathways.     

Expression,purification and spectroscopic characterization of the cytochrome P450 CYP121 from Mycobacterium tuberculosis

The CYP121 gene from the pathogenic bacterium Mycobacterium tuberculosis has been cloned and expressed in Escherichia coli, and the protein purified to homogeneity by ion exchange and hydrophobic interaction chromatography. The CYP121 gene encodes a cytochrome P450 enzyme (CYP121) that displays typical electronic absorption features for a member of this superfamily of hemoproteins (major Soret absorption band at 416.5 nm with alpha and beta bands at 565 and 538 nm, respectively, in the oxidized form) and which binds carbon monoxide to give the characteristic Soret band shift to 448 nm. Resonance Raman, EPR and MCD spectra show the protein to be predominantly low-spin and to have a typical cysteinate- and water-ligated b-type heme iron. CD spectra in the far UV region describe a mainly alpha helical conformation, but the visible CD spectrum shows a band of positive sign in the Soret region, distinct from spectra for other P450s recognized thus far. CYP121 binds very tightly to a range of azole antifungal drugs (e.g. clotrimazole, miconazole), suggesting that it may represent a novel target for these antibiotics in the M. tuberculosis pathogen.     

Identification of variable amino acids in the SRS1 region of CYP6B1 modulating furanocoumarin metabolism

The homology model of Papilio polyxenes CYP6B1 places Ile115, one of two variable amino acids, in the SRS1 of various CYP6B subfamily proteins in close proximity to the heme and Ala113, another variable amino acid, in a more distal position. We have constructed mutant CYP6B1 proteins altered at either of these positions and homology models of each based on multiple alignments with crystallized P450 proteins. The homology models suggest the existence of significant structural diversity in the hydrogen bond network surrounding the heme as a result of single point mutations in SRS1. Mutagenesis of Ile115 or Ala113 to other residues present in the insect CYP6B subfamily indicates that these amino acids control the spin state of the heme and, as a result, the catalytic activity of this monooxygenase. In particular, the I115L mutation significantly increases the spin state of the heme coordinately with 2- to 4-fold increases in its turnover of linear furanocoumarins. Other A113V, A113L, A113Q, and A113E mutations display more variation in their effects but, in each case, strong correlations exist between furanocoumarin turnover and heme spin state. These data demonstrate that variable amino acids in SRS1 of the insect CYP6B subfamily exert dramatic effects on the range of furanocoumarins metabolized, even when they occur in positions potentially distal from the substrate. These effects are possibly mediated through rearrangement of the local hydrogen bond network.