Ultrahigh resolution structures of nitrophorin 4: heme distortion in ferrous CO and NO complexes

Nitrophorin 4 (NP4), a nitric oxide (NO)-transport protein from the blood-sucking insect Rhodnius prolixus, uses a ferric (Fe3+) heme to deliver NO to its victims. NO binding to NP4 induces a large conformational change and complete desolvation of the distal pocket. The heme is markedly nonplanar, displaying a ruffling distortion postulated to contribute to stabilization of the ferric iron. Here, we report the ferrous (Fe2+) complexes of NP4 with NO, CO, and H2O formed after chemical reduction of the protein and the characterization of these complexes by absorption spectroscopy, flash photolysis, and ultrahigh-resolution crystallography (resolutions vary from 0.9 to 1.08 A). The absorption spectra, both in solution and in the crystal, are typical for six-coordinated ferrous complexes. Closure and desolvation of the distal pocket occurs upon binding CO or NO to the iron regardless of the heme oxidation state, confirming that the conformational change is driven by distal ligand polarity. The degree of heme ruffling is coupled to the nature of the ligand and the iron oxidation state in the following order: (Fe3+)-NO > (Fe2+)-NO > (Fe2+)-CO > (Fe3+)-H2O > (Fe2+)-H2O. The ferrous coordination geometry is as expected, except for the proximal histidine bond, which is shorter than typically found in model compounds. These data are consistent with heme ruffling and coordination geometry serving to stabilize the ferric state of the nitrophorins, a requirement for their physiological function. Possible roles for heme distortion and NO bending in heme protein function are discussed.     

Breaking the proximal Fe(II)-N(His) bond in heme proteins through local structural tension: lessons from the heme b proteins nitrophorin 4, nitrophorin 7, and related site-directed mutant proteins

The factors leading to the breakage of the proximal iron-histidine bond in the ferroheme protein soluble guanylate cyclase (sGC) are still a matter of debate. This event is a key mechanism in the sensing of NO that leads to the production of the second-messenger molecule cGMP. Surprisingly, in the heme protein nitrophorin 7 (NP7), we noticed by UV-vis absorbance spectroscopy and resonance Raman spectroscopy that heme reduction leads to a loss of the proximal histidine coordination, which is not observed for the other isoproteins (NP1-4). Structural considerations led to the generation and spectroscopic investigation of site-directed mutants NP7(E27V), NP7(E27Q), NP4(D70A), and NP2(V24E). Spectroscopic investigation of these proteins shows that the spatial arrangement of residues Glu27, Phe43, and His60 in the proximal heme pocket of NP7 is the reason for the weakened Fe(II)-His60 bond through steric demand. Spectroscopic investigation of the sample of NP7 reconstituted with 2,4-dimethyldeuterohemin ("symmetric heme") demonstrated that the heme vinyl substituents are also responsible. Whereas the breaking of the iron-histidine bond is rarely seen among unliganded ferroheme proteins, the breakage of the Fe(II)-His bond upon binding of NO to the sixth coordination site is sometimes observed because of the negative trans effect of NO. However, it is still rare among the heme proteins, which is in contrast to the case for trans liganded nitrosyl model hemes. Thus, the question of which factors determine the Fe(II)-His bond labilization in proteins arises. Surprisingly, mutant NP2(V24E) turned out to be particularly similar in behavior to sGC; i.e., the Fe(II)-His bond is sensitive to breakage upon NO binding, whereas the unliganded form binds the proximal His at neutral pH. To the best of our knowledge, NP2(V24E) is the first example in which the ability to use the His-on ? His-off switch was engineered into a heme protein by site-directed mutagenesis other than the proximal His itself. Steric tension is, therefore, introduced as a potential structural determinant for proximal Fe(II)-His bond breakage in heme proteins.     

Structural dynamics controls nitric oxide affinity in nitrophorin 4

Nitrophorin 4 (NP4) is one of seven nitric oxide (NO) transporting proteins in the blood-sucking insect Rhodnius prolixus. In its physiological function, NO binds to a ferric iron centered in a highly ruffled heme plane. Carbon monoxide (CO) also binds after reduction of the heme iron. Here we have used Fourier transform infrared spectroscopy at cryogenic temperatures to study CO and NO binding and migration in NP4, complemented by x-ray cryo-crystallography on xenon-containing NP4 crystals to identify cavities that may serve as ligand docking sites. Multiple infrared stretching bands of the heme-bound ligands indicate different active site conformations with varying degrees of hydrophobicity. Narrow infrared stretching bands are observed for photodissociated CO and NO; temperature-derivative spectroscopy shows that these bands are associated with ligand docking sites close to the extremely reactive heme iron. No rebinding from distinct secondary sites was detected, although two xenon binding cavities were observed in the x-ray structure. Photolysis studies at approximately 200 K show efficient NO photoproduct formation in the more hydrophilic, open NP4 conformation. This result suggests that ligand escape is facilitated in this conformation, and blockage of the active site by water hinders immediate reassociation of NO to the ferric iron. In the closed, low-pH conformation, ligand escape from the active site of NP4 is prevented by an extremely reactive heme iron and the absence of secondary ligand docking sites.     

Replacement of the axial histidine heme ligand with cysteine in nitrophorin 1: spectroscopic and crystallographic characterization

To evaluate the potential of using heme-containing lipocalin nitrophorin 1 (NP1) as a template for protein engineering, we have replaced the native axial heme-coordinating histidine residue with glycine, alanine, and cysteine. We report here the characterization of the cysteine mutant H60C_NP1 by spectroscopic and crystallographic methods. The UV/vis, resonance Raman, and magnetic circular dichroism spectra suggest weak thiolate coordination of the ferric heme in the H60C_NP1 mutant. Reduction to the ferrous state resulted in loss of cysteine coordination, while addition of exogenous imidazole ligands gave coordination changes that varied with the ligand. Depending on the substitution of the imidazole, we could distinguish three heme coordination states: five-coordinate monoimidazole, six-coordinate bisimidazole, and six-coordinate imidazole/thiolate. Ligand binding affinities were measured and found to be generally 2–3 orders of magnitude lower for the H60C mutant relative to NP1. Two crystal structures of the H60C_NP1 in complex with imidazole and histamine were solved to 1.7- and 1.96-Å resolution, respectively. Both structures show that the H60C mutation is well tolerated by the protein scaffold and suggest that heme–thiolate coordination in H60C_NP1 requires some movement of the heme within its binding cavity. This adjustment may be responsible for the ease with which the engineered heme–thiolate coordination can be displaced by exogenous ligands.     

Assignment of heme resonances and determination of the electronic structures of high- and low-spin nitrophorin 2 by 1H and 13C NMR spectroscopy: an explanation of the order of heme methyl resonances in high-spin ferriheme proteins

The (1)H NMR resonances of the heme substituents of the low-spin Fe(III) form of nitrophorin 2, as its complexes with N-methylimidazole (NP2-NMeIm) and imidazole (NP2-ImH), have been assigned by a combination of (1)H homonuclear two-dimensional NMR techniques and (1)H-(13)C HMQC. Complete assignment of the proton and partial assignment of the (13)C resonances of the heme of these complexes has been achieved. Due to favorable rates of ligand exchange, it was also possible to assign part of the (1)H resonances of the high-spin heme via saturation transfer between high- and low-spin protein forms in a partially liganded NP2-NMeIm sample; additional resonances (vinyl and propionate) were assigned by NOESY techniques. The order of heme methyl resonances in the high-spin form of the protein over the temperature range of 10-37 degrees C is 8 = 5 > 1 > 3; the NMeIm complex has 5 > 1 > 3 > 8 as the order of heme methyl resonances at <30 degrees C, while above that temperature, the order is 5 > 3 > 1 > 8, due to crossover of the closely spaced 3- and 1-methyl resonances of the low-spin complex at higher temperatures. This crossover defines the nodal plane of the heme orbital used for spin delocalization as being oriented 162 +/- 2 degrees clockwise from the heme N(II)-Fe-N(IV) axis for the heme in the B orientation. For the NP2-ImH complex, the order of heme methyl resonances is 3 > 5 > 1 > 8, which defines the orientation of the nodal plane of the heme orbital used for spin delocalization as being oriented approximately 150-155 degrees clockwise from the heme N(II)-Fe-N(IV) axis. In both low-spin complexes, the results are most consistent with the exogenous planar ligand controlling the orientation of the nodal plane of the heme orbital. In the high-spin form of NP2, the proximal histidine plane is shown to be oriented 135 degrees clockwise from the heme N(II)-Fe-N(IV) axis, again for the B heme orientation. A correlation between the order of heme methyl resonances in the high-spin form of NP2 and several other ferriheme proteins and an apparent 90 degrees shift in the nodal plane of the orbital involved in spin delocalization from that expected on the basis of the orientation of the axial histidine imidazole nodal plane have been explained in terms of bonding interactions between Fe(III), the axial histidine imidazole nitrogen, and the porphyrin pi orbitals of the high-spin protein.     

Reduction of the lipocalin type heme containing protein nitrophorin -- sensitivity of the fold-stabilizing cysteine disulfides toward routine heme-iron reduction

The determination of the redox properties of the cofactor in heme proteins provides fundamental insight into the chemical characteristics of this wide-spread class of metalloproteins. For the preparation of the ferroheme state, probably the most widely applied reductant is sodium dithionite, which at neutral pH has a reduction potential well below the reduction potential of most heme centers. In addition to the heme iron, some heme proteins, including the nitrophorins (NPs), contain cysteinecysteine disulfide bonds. In the present study, the effect of dithionite on the disulfides of NP4 and NP7 is addressed. To gain deeper understanding of the disulfide/dithionite reaction, oxidized glutathione (GSSG), as a model system, was incubated with dithionite and the products were characterized by ¹³C NMR spectroscopy and reverse phase chromatography in combination with mass spectrometry. This revealed the formation of one equivalent each of thiol (GSH) and glutathione-S-thiosulfate (GSSO₃⁻). With this background information, the effect of dithionite on the cystines of NP4 and NP7 was studied after trapping of the thiols with para-cloromercurybenzyl sulfonate (p-CMBS) and subsequent matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) where the heterolytic cleavage of the SS bond appears with only 2 molar equivalents of the reductant. Furthermore, prolonged electrochemical reduction of NP4 and NP7 in the presence of electrochemical mediators also leads to disulfide breakage. However, due to sterical shielding of the disulfide bridges in NP4 and NP7, the cystine reduction can be largely prevented by the use of stoichiometric amounts of reductant or limited electrochemical reduction. The described disulfide breakage during routine iron reduction is of importance for other heme proteins containing cystine(s).     

Resonance Raman spectra of the heme in leghemoglobin. Evidence for the absence of ruffling and the influence of the vinyl groups

Resonance Raman spectra of deoxy and carbonmonoxy leghemoglobin (Lb) are compared to the corresponding forms of human adult hemoglobin (HbA). It is found that the heme "core size" indicator line has nearly the same frequency for the two deoxyhemoglobins and the pi-electron density-sensitive line also falls at the same frequency. However, several other modes occur at very different frequencies in the spectra of the two proteins. From an examination of the spectrum of an HbA derivative in which the beta-carbon atoms of the heme vinyl groups were deuterated, it appears that the major differences between deoxy-HbA and -Lb may result from conformational changes in the vinyl groups. No evidence for the suggested ruffling (Irwin, M. J., Armstrong, R. S., and Wright, P. E. (1981) FEBS Lett. 133, 239-243) in deoxy-Lb was found. The spectra of carbonmonoxy-Lb and -HbA were also found to be very different. As in the deoxy case, some of these frequency differences could be attributed to vinyl group conformational differences. However, from the large difference in the pi-electron density-sensitive line, it appears that the vinyl pi-conjugation into the porphyrin in Lb(CO) may be different than it is in HbA(CO). The vinyl conformational differences may be a consequence of the looser heme pocket in Lb than in HbA. The difference in pi-conjugation could make a significant contribution to the difference in ligand binding affinity for these two globins.     

Coordination geometry of heme in lactoperoxidase: pH-dependent 1H relaxivity and optical spectral studies

Molar relaxivity of water proton in lactoperoxidase solution was studied as a function of pH in the range of 2-13 by spin-lattice relaxation time measurements on a Bruker AM 500 MHz nuclear magnetic resonance (NMR) spectrometer. It was shown by comparison with the molar relaxivities of met myoglobin (Mb) and horseradish peroxidase (HRP) solutions that the sixth coordination position of the heme pocket in lactoperoxidase (LPO) is vacant. Distance of the water proton in the heme pocket from ferric ion was deduced to be 2.7, 3.6 and 4.3 A for Mb, HRP, and LPO, respectively. Acid-alkaline transition for met myoglobin, horseradish peroxidase, and lactoperoxidase determined from the pH dependence of changes in the Soret absorptions were found to be characterized by pK of 8.8, 10.9, and 12.1, respectively. Proton NMR of LPO at pH = 12.2 was found to have single broad resonance considerably upfield shifted as compared to that of LPO at neutral pH. By comparison with the proton NMR of HRP and Mb at pH greater than their respective pK of acid-alkaline transition, the upfield shifted proton resonance of LPO at pH = 12.2 was assigned to be due to low-spin LPO.     

NMR studies of nitrophorin distal pocket side chain effects on the heme orientation and seating of NP2 as compared to NP1

The nitrophorins (NP) of the adult blood-sucking insect Rhodnius prolixus fall into two pairs based on sequence identity (NP1,4 (90%) and NP2,3 (79%)), which differ significantly in the size of side chains of residues which contact the heme. These residues include those in the distal pocket of NP2 (I120) and NP1 (T121) and the “belt” that surrounds the heme of NP2 (S40, F42), and NP1(A42, L44). To determine the importance of these residues and others conserved or very similar for the two pairs, including L122(123), L132(133), appropriate mutants of NP2 and NP1 have been prepared and studied by ¹H NMR spectroscopy. Wild-type NP2 has heme orientation ratio (A:B) of 1:8 at equilibrium, while wild-type NP1 has A:B ~ 1:1 at equilibrium. Another difference between NP2 and NP1 is in the heme seating with regard to His57(59). It is found that among the distal pocket residues investigated, the residue most responsible for heme orientation and seating is I120(T121). F42(L44) and L106(F107) may also be important, but must be investigated in greater detail.     

The carbohydrate-binding site in galectin-3 is preorganized to recognize a sugarlike framework of oxygens: ultra-high-resolution structures and water dynamics

The recognition of carbohydrates by proteins is a fundamental aspect of communication within and between living cells. Understanding the molecular basis of carbohydrate-protein interactions is a prerequisite for the rational design of synthetic ligands. Here we report the high- to ultra-high-resolution crystal structures of the carbohydrate recognition domain of galectin-3 (Gal3C) in the ligand-free state (1.08 ? at 100 K, 1.25 ? at 298 K) and in complex with lactose (0.86 ?) or glycerol (0.9 ?). These structures reveal striking similarities in the positions of water and carbohydrate oxygen atoms in all three states, indicating that the binding site of Gal3C is preorganized to coordinate oxygen atoms in an arrangement that is nearly optimal for the recognition of β-galactosides. Deuterium nuclear magnetic resonance (NMR) relaxation dispersion experiments and molecular dynamics simulations demonstrate that all water molecules in the lactose-binding site exchange with bulk water on a time scale of nanoseconds or shorter. Nevertheless, molecular dynamics simulations identify transient water binding at sites that agree well with those observed by crystallography, indicating that the energy landscape of the binding site is maintained in solution. All heavy atoms of glycerol are positioned like the corresponding atoms of lactose in the Gal3C complexes. However, binding of glycerol to Gal3C is insignificant in solution at room temperature, as monitored by NMR spectroscopy or isothermal titration calorimetry under conditions where lactose binding is readily detected. These observations make a case for protein cryo-crystallography as a valuable screening method in fragment-based drug discovery and further suggest that identification of water sites might inform inhibitor design.     

Effect of the N-terminus on heme cavity structure, ligand equilibrium, rate constants, and reduction potentials of nitrophorin 2 from Rhodnius prolixus

The D1A mutant of recombinant NP2 has been prepared and shown to have the expression-initiation methionine-0 cleaved during expression in E. coli, as is the case for recombinant NP4, where Ala is the first amino acid for the recombinant protein as well as for the mature native protein. The heme substituent 1H NMR chemical shifts of NP2-D1A and those of its imidazole, N-methylimidazole, and cyanide complexes are rather different from those of NP2-M0D1. This difference is likely due to the much smaller size of the N-terminal amino acid (A) of NP2-D1A, which allows the formation of the closed loop form of this protein, as it does for NP4 (Weichsel, A., Andersen, J. F., Roberts, S. A., and Montfort, W. R. (2000) Nature Struct. Biol. 7, 551-554). The ratio of the two hemin rotational isomers A and B is different for the two proteins, and the rate at which the A:B ratio reaches equilibrium is strikingly different (NP2-M0D1 t1/2 for heme rotation approximately 2 h, NP2-D1A t1/2 approximately 43 h). This difference is consistent with the high stability of the closed loop form of the NP2-D1A protein and infrequent opening of the loops that could allow heme to at least partially exit the binding pocket in order to rotate about its alpha,gamma-meso axis. Consistent with this, the rates of histamine binding and release to/from NP2-D1A are significantly slower than those for NP2-M0D1 at pH 7.5. This work suggests that care must be taken in interpreting data obtained from proteins that carry the expression-initiation M0.     

Ligand-induced conformational changes in the crystal structures of Pneumocystis carinii dihydrofolate reductase complexes with folate and NADP+

Structural data from two independent crystal forms (P212121 and P21) of the folate (FA) binary complex and from the ternary complex with the oxidized coenzyme, NADP+, and recombinant Pneumocystis carinii dihydrofolate reductase (pcDHFR) refined to an average of 2.15 A resolution, show the first evidence of ligand-induced conformational changes in the structure of pcDHFR. These data are also compared with the crystal structure of the ternary complex of methotrexate (MTX) with NADPH and pcDHFR in the monoclinic lattice with data to 2.5 A resolution. Comparison of the data for the FA binary complex of pcDHFR with those for the ternary structures reveals significant differences, with a >7 A movement of the loop region near residue 23 that results in a new "flap-open" position for the binary complex, and a "closed" position in the ternary complexes, similar to that reported for Escherichia coli (ec) DHFR complexes. In the orthorhombic lattice for the binary FA pcDHFR complex, there is also an unwinding of a short helical region near residue 47 that places hydrophobic residues Phe-46 and Phe-49 toward the outer surface, a conformation that is stabilized by intermolecular packing contacts. The pyrophosphate moiety of NADP+ in the ternary folate pcDHFR complexes shows significant differences in conformation compared with that observed in the MTX-NADPH-pcDHFR ternary complex. Additionally, comparison of the conformations among these four pcDHFR structures reveals evidence for subdomain movement that correlates with cofactor binding states. The larger binding site access in the new "flap-open" loop 23 conformation of the binary FA complex is consistent with the rapid release of cofactor from the product complex during catalysis as well as the more rapid release of substrate product from the binary complex as a result of the weaker contacts of the closed loop 23 conformation, compared to ecDHFR.     

Fractal geometry and root system structures of heterogeneous plant communities

Above-ground plant growth is widely known in terms of structural diversity. Likewise, the below-ground growth presents a mosaic of heterogeneous structures of differing complexity. In this study, root system structures of heterogeneous plant communities were recorded as integral systems by using the trench profile method. Fractal dimensions of the root images were calculated from image files by the box-counting method. This method allows the structural complexity of such associations to be compared between plant communities, with regard to their potentials for soil resource acquisition and utilization. Distinct and partly significant differences are found (fractal dimension between 1.46±0.09 and 1.71±0.05) in the below-ground structural complexity of plant communities, belonging to different biotope types. The size of the heterogeneous plant community to be examined has an crucial influence on the fractal dimension of the root system structures. The structural heterogeneity becomes particularly evident (fractal dimensions between 1.32 and 1.77) when analysing many small units of a complex root system association. In larger plant communities, a broad variety of below-ground structures is recorded in its entirety, integrating the specific features of single sub-structures. In that way, extreme fractal dimensions are lost and the diversity decreases. Therefore, the analysis of larger units of root system associations provides a general knowledge of the complexity of root system structures for heterogeneous plant communities.     

NMR study of hybrid hemoglobins containing unnatural heme: effect of heme modification on their tertiary and quaternary structures

The effect of heme modification on the tertiary and quaternary structures of hemoglobins was examined by utilizing the NMR spectra of the reconstituted [mesohemoglobin (mesoHb), deuterohemoglobin (deuteroHb)] and hybrid heme (meso-proto, deutero-proto) hemoglobins (Hbs). The heme peripheral modification resulted in the preferential downfield shift of the proximal histidine N1H signal for the beta subunit, indicating nonequivalence of the structural change induced by the heme modification in the alpha and beta subunits of Hb. In the reconstituted and hybrid heme Hbs, the exchangeable proton resonances due to the intra- and intersubunit hydrogen bonds, which have been used as the oxy and deoxy quaternary structural probes, were shifted by 0.2-0.3 ppm from that of native Hb upon the beta-heme substitution. This suggests that, in the fully deoxygenated form, the quaternary structure of the reconstituted Hbs is in an "imperfect" T state in which the hydrogen bonds located at the subunit interface are slightly distorted by the conformational change of the beta subunit. Moreover, the two heme orientations are found in the alpha subunit of deuteroHb, but not in the beta subunit of deuteroHb, and in both the alpha and beta subunits of mesoHb. The tertiary and quaternary structural changes in the Hb molecule induced by the heme peripheral modification were also discussed in relation to their functional properties.     

Role of binding site loops in controlling nitric oxide release: structure and kinetics of mutant forms of nitrophorin 4

Nitrophorins are ferric heme proteins that transport nitric oxide (NO) from blood-sucking insects to victims. NO binding is tighter at lower pH values, as found in the insect salivary gland, and weaker at the pH of the victim's tissue, facilitating NO release and subsequent vasodilation. Previous structural analyses of nitrophorin 4 (NP4) from Rhodnius prolixus revealed a substantial NO-induced conformational change involving the A-B and G-H loops, which rearrange to desolvate the distal pocket and pack nonpolar residues against the heme-ligated NO. Previous kinetic analyses revealed a slow, biphasic, and pH-dependent NO release, which was proposed to be associated with loop movements. In this study, we created NP4 mutants D30A and D30N (A-B loop), D129A/L130A (G-H loop), and T121V (distal pocket). Eight crystal structures were determined, including complexes with NO, NH(3), and imidazole, to resolutions as high as 1.0 A. The NO-induced conformational change is largely abolished in the loop mutants, but retained in T121V. Kinetic analyses using stopped-flow spectroscopy revealed the pH dependence for NO release is eliminated for D129A/L130A, considerably reduced for D30A and D30N, but retained for T121V. NO association rates were increased 2-5-fold for T121V, but were unchanged in the loop mutants. Taken together, our findings demonstrate that the pH dependency for NO release is linked to loop dynamics and that solvent reorganization is apparently rate-limiting for formation of the initial iron-nitrosyl bond. Interestingly, the multiphasic kinetic behavior of rNPs was not affected by mutations, and its cause remains unclear.     

Random cloning of bent DNA segments from Saccharomyces cerevisiae and primary characterization of their structures

Summary Until recently it was assumed that any short segment of DNA could be approximated as a straight rod. Many instances, however, have been reported in which the helical axis is curved. We have devised a simple method for selective identification of DNA segments containing a sequence-directed bend (curvature), by means of a two-dimensional polyacrylamide gel electrophoresis. In order to gain general insights into the structural features and the functional significance of sequence-directed bends, a bank of plasmids carrying bent DNA inserts from the Saccharomyces cerevisiae total genomic DNA was constructed. Primary characterizations of a set of bent DNA segments randomly cloned from S. cerevisiae are presented. One of the cloned DNA segments appears to be derived from a yeast plasmid, the 2 m circle DNA.     

Alteration of P450 distal pocket solvent leads to impaired proton delivery and changes in heme geometry

Distal pocket water molecules have been widely implicated in the delivery of protons required in O-O bond heterolysis in the P450 reaction cycle. Targeted dehydration of the cytochrome P450cam (CYP101) distal pocket through mutagenesis of a distal pocket glycine to either valine or threonine results in the alteration of spin state equilibria, and has dramatic consequences on the catalytic rate, coupling efficiency, and kinetic solvent isotope effect parameters, highlighting an important role of the active-site hydration level on P450 catalysis. Cryoradiolysis of the mutant CYP101 oxyferrous complexes further indicates a specific perturbation of proton-transfer events required for the transformation of ferric-peroxo to ferric-hydroperoxo states. Finally, crystallography of the 248Val and 248Thr mutants in both the ferric camphor bound resting state and ferric-cyano adducts shows both the alteration of hydrogen-bonding networks and the alteration of heme geometry parameters. Taken together, these results indicate that the distal pocket microenvironment governs the transformation of reactive heme-oxygen intermediates in P450 cytochromes.