Scanning chimeragenesis: the approach used to change the substrate selectivity of fatty acid monooxygenase CYP102A1 to that of terpene omega-hydroxylase CYP4C7.

CYP102A1 is a highly active, water-soluble, bacterial monooxygenase enzyme that contains both substrate-binding heme and diflavin reductase subunits, both in a single polypeptide. Recently we developed a procedure which uses the known structure of the substrate-bound heme domain of CYP102A1 and its sequence homology with a cytochrome P450 of unknown structure, both of which react with a common substrate but produce different products, to create recombinant enzymes which have substrate selectivity different from that of CYP102A1, and produce the product of the enzyme of unknown structure. Insect CYP4C7, a terpene hydroxylase from the cockroach, was chosen as the cytochrome P450 of unknown structure, and farnesol was chosen as the substrate. CYP102A1 oxidizes farnesol to three products (2,3-epoxyfarnesol, 10,11-epoxyfarnesol, and 9-hydroxyfarnesol), whereas CYP4C7 produces 12-hydroxyfarnesol as the major product. In earlier work it was found that the chimera C(78-82,F87L) showed a change in substrate selectivity from fatty acids to farnesol, and was approximately sixfold more active than wild-type CYP102A1 (Chen et al. in J Biol Inorg Chem 13:813-824, 2008), but neither it nor any other earlier chimera produced 12-hydroxyfarnesol. In this work we added amino acid residues 327-332, to create six new full-length, functional chimeric proteins. Four of these, the most active of which was C(78-82,F87L,328-330), produce 12-hydroxyfarnesol as the major product, with approximately twofold increase in turnover number as compared with wild-type CYP102A1 toward farnesol. Methylfarnesoate was metabolized to 12-hydroxymethylfarnesoate (70%) and 10,11-epoxymethylfarnesoate (juvenile hormone III) (30%). The latter is metabolized to 65% 12-hydroxy-10,11-epoxymethylfarnesoate and 35% 15-hydroxy-10,11-epoxymethylfarnesoate. Substitution of residues 328-330, APA, by VPL was crucial to accomplishing this change in product.

NMR and EPR studies of chloroiron(III) tetraphenyl-chlorin and its complexes with imidazoles and pyridines of widely differing basicities.

The NMR and EPR spectra of two bisimidazole and three bispyridine complexes of tetraphenylchlorinatoiron(III), [(TPC)Fe(L)2]+ (L = Im-d4, 2-MeHIm, 4-Me2NPy, Py, and 4-CNPy), have been investigated. The full resonance assignments of the [(TPC)Fe(L)2]+ complexes of this study have been made from correlation spectroscopy (COSY) and nuclear Overhauser enhancement spectroscopy (NOESY) experiments and Amsterdam density functional (ADF) calculations. Unlike the [(OEC)Fe(L)2]+ complexes reported previously (Cai, S.; Lichtenberger, D. L.; Walker, F. A. Inorg. Chem. 2005, 44, 1890-1903), the NMR data for the [(TPC)Fe(L)2]+ complexes of this study indicate that the ground state is S = 1/2 for each bisligand complex, whereas a higher spin state was present at NMR temperatures for the Py and 4-CNPy complexes of (OEC)Fe(III). The pyrrole-8,17 and pyrroline-H of all [TPCFe(L)2]+ show large magnitude chemical shifts (hence indicating large spin density on the adjacent carbons that are part of the pi system), while pyrrole-12,13-CH2 and -7,18-CH2 protons show much smaller chemical shifts, as predicted by the spin densities obtained from ADF calculations. The magnitude of the chemical shifts decreases with decreasing donor ability of the substituted pyridine ligands, with the nonhindered imidazole ligand having slightly larger magnitude chemical shifts than the most basic pyridine, even though its basicity is significantly lower (4-Me2NPyH+ pKa = 9.7, H2Im+ pKa = 6.65 (adjusted for the statistical factor of 2 protons)). The temperature dependence of the chemical shifts of all but the 4-Me2NPy bisligand complexes studied over the temperature range of the NMR investigations shows that they have mixed (dxy)2(dxz,dyz)3/(dxzdyz)4(dxy)1 electron configurations that cannot be resolved by temperature-dependent fitting of the proton chemical shifts, with an S = 3/2 excited state in each case that in most cases lies at more than kT at room temperature above the ground state. The observed pattern of chemical shifts of the 4-CNPy complex and analysis of the temperature dependence indicate that it has a pure (dxzdyz)4(dxy)1 ground state and that it is ruffled, because ruffling mixes the a(2u)(pi)-like orbital of the chlorin into the singly occupied molecular orbital (SOMO). This mixing accounts for the negative chemical shift of the pyrroline-H (-6.5 ppm at -40 degrees C) and thus the negative spin density at the pyrroline-alpha-carbons, but the mixing is not to the same extent as observed for [(TPC)Fe(t-BuNC)2]+, whose pyrroline-H chemical shift is -36 ppm at 25 degrees C (Simonneaux, G.; Kobeissi, M. J. Chem. Soc., Dalton Trans. 2001, 1587-1592). Peak assignments for high-spin (TPC)FeCl have been made by saturation transfer techniques that depend on chemical exchange between this complex and its bis-4-Me2NPy adduct.

Heme-assisted S-nitrosation of a proximal thiolate in a nitric oxide transport protein.

Certain bloodsucking insects deliver nitric oxide (NO) while feeding, to induce vasodilation and inhibit blood coagulation. We have expressed, characterized, and determined the crystal structure of the Cimex lectularius (bedbug) nitrophorin, the protein responsible for NO storage and delivery, to understand how the insect successfully handles this reactive molecule. Surprisingly, NO binds not only to the ferric nitrophorin heme, but it can also be stored as an S-nitroso (SNO) conjugate of the proximal heme cysteine (Cys-60) when present at higher concentrations. EPR- and UV-visible spectroscopies, and a crystallographic structure determination to 1.75-A resolution, reveal SNO formation to proceed with reduction of the heme iron, yielding an Fe-NO complex. Stopped-flow kinetic measurements indicate that an ordered reaction mechanism takes place: initial NO binding occurs at the ferric heme and is followed by heme reduction, Cys-60 release from the heme iron, and SNO formation. Release of NO occurs through a reversal of these steps. These data provide, to our knowledge, the first view of reversible metal-assisted SNO formation in a protein and suggest a mechanism for its role in NO release from ferrous heme. This mechanism and Cimex nitrophorin structure are completely unlike those of the nitrophorins from Rhodnius prolixus, where NO protection is provided by a large conformational change that buries the heme nitrosyl complex, highlighting the remarkable evolution of proteins that assist insects in bloodfeeding.

Axial ligand complexes of the Rhodnius nitrophorins: reduction potentials, binding constants, EPR spectra, and structures of the 4-iodopyrazole and imidazole complexes of NP4.

Previously, we utilized 4-iodopyrazole (4IPzH) as a heavy atom derivative for the initial solution of the crystal structure of the nitrophorin from Rhodnius prolixus, NP1, where it was found to bind to the heme with the iodo group disordered in two positions. We have now determined the structure of the 4IPzH complex of NP4 at pH 7.5 and find that the geometry and bond lengths at the iron center are extremely similar to those of the imidazole (ImH) complex of the same protein (structure determined at pH 5.6), except that the G-H loop is not in the closed conformation. 4IPzH binds to the heme of NP4 in an ordered manner, with the iodo substituent pointed toward the opening of the heme pocket, near the surface of the protein. In order to understand the solution chemistry in terms of the relative binding abilities of 4IPzH, ImH, and histamine (Hm, a physiological ligand for the nitrophorins), we have also investigated the equilibrium binding constants and reduction potentials of these three ligand complexes of the four Rhodnius nitrophorins as a function of pH. We have found that, unlike the other Lewis bases, 4IPzH forms less stable complexes with the Fe(III) than the Fe(II) oxidation states of NP1 and NP4, and similar stability for the two oxidation states of NP2 and NP3, suggesting that this ligand is a softer base than ImH or Hm, for both of which the Fe(III) complexes are more stable than those of Fe(II) for all four nitrophorins. Surprisingly, in spite of this and the much lower basicity of 4IPzH than imidazole and histamine, the EPR g-values of all three ligand complexes are very similar.

Models of the bis-histidine-coordinated ferricytochromes: Mössbauer and EPR spectroscopic studies of low-spin iron(III) tetrapyrroles of various electronic ground states and axial ligand orientations.

The EPR and magnetic Mössbauer spectra of a series of axial ligand complexes of tetrakis(2,6-dimethoxyphenyl)porphyrinatoiron(III), [(2,6-(OMe)(2))(4)TPPFeL(2)](+), where L= N-methylimidazole, 2-methylimidazole, or 4-(dimethylamino)pyridine, of one axial ligand complex of tetraphenylporphyrin, the bis(4-cyanopyridine) complex [TPPFe(4-CNPy)(2)](+), and of one axial ligand complex of tetraphenylchlorin, [TPCFe(ImH)(2)](+), where ImH=imidazole, have been investigated and compared to those of low-spin Fe(III) porphyrinates and ferriheme proteins reported in the literature. On the basis of this and previous complementary spectroscopic investigations, three types of complexes have been identified: those having (d(xy))(2)(d(xz),d(yz))(3) electronic ground states with axial ligands aligned in perpendicular planes (Type I), those having (d(xy))(2)(d(xz),d(yz))(3) electronic ground states with axial ligands aligned in parallel planes (Type II), and those having the novel (d(xz),d(yz))(4)(d(xy))(1) electronic ground state (Type III). A subset of the latter type, with planar axial ligands aligned parallel to each other or strong macrocycle asymmetry that yield rhombic EPR spectra, cannot be created using the porphyrinate ligand. Type I centers are characterized by "large g(max)" EPR spectra with g>3.2 and well-resolved, widely spread magnetic Mössbauer spectra having A(zz)/ g(N)mu(N)>680 kG, with A(xx) negative in sign but much smaller in magnitude than A(zz), while Type II centers have well-resolved rhombic EPR spectra with g(zz)=2.4-3.1 and also less-resolved magnetic Mössbauer spectra, and usually have A(zz)/ g(Nmu(N) in the range of 440-660 kG (but in certain cases as small as 180 kG) and A(xx) again negative in sign but only somewhat smaller (but occasionally larger in magnitude) than A(zz), and Type III centers have axial EPR spectra with g( upper left and right quadrants ) approximately 2.6 or smaller and g( vertical line )<1.0-1.95, but often not resolved, and less-resolved magnetic Mössbauer spectra having A(zz)/ g(N)mu(N) in the range of 270-400 kG, and A(xx) again negative in sign but much smaller in magnitude than A(zz). An exception to this rule is [TPPFe(4-CNPy)(2)](+), which has A(xx)/ g(N)mu(N)=-565 kG, A(yy)/ g(N)mu(N)=629 kG, and A(zz)/ g(N)mu(N)=4 kG. A subset of Type II complexes (Type II') have rhombicities ( V/Delta) much greater than 0.67 and A(zz)/ g(N)mu(N) ranging from 320 to 170 kG, with A(xx) also negative but with the magnitude of A(xx) significantly larger than that of A(zz). These classifications are also observed for a variety of ferriheme proteins, and they lead to linear correlations between A(zz) and either A(xx), g(zz), or V/Delta for Types I and II (but not for A(zz) versus V/Delta for Type II'). Not enough data are yet available on Type III complexes to determine what, if any, correlations may be observed.

Electrochemical and NMR spectroscopic studies of distal pocket mutants of nitrophorin 2: stability, structure, and dynamics of axial ligand complexes.

WT and leucine --> valine distal pocket mutants of nitrophorin 2 (NP2) and their NO complexes have been investigated by spectroelectrochemistry. NO complexes of two of the mutants exhibit more positive reduction potential shifts than does the WT protein, thus indicating stabilization of the Fe(II)-NO state. This more positive reduction potential for NP2-L132V and the double mutant is consistent with the hypothesis that smaller valine residues may allow the heme to regain planarity instead of being significantly ruffled, as in WT NP2. Thus, ruffling may stabilize the Fe(III)-NO state, which is required for facile NO dissociation. NMR spectroscopic investigations show that the sterically demanding 2-methylimidazole ligand readily binds to all three distal pocket mutants to create low-spin Fe(III) complexes having axial ligands in nearly perpendicular planes; it also binds to the WT protein in the presence of higher concentrations of 2-methylimidazole, but yields a different ligand plane orientation than is present in any of the three distal pocket mutants. NOESY spectra of NP2-ImH mutants exhibit chemical exchange cross peaks, whereas WT NP2-ImH shows no chemical exchange. Chemical exchange in the case of the distal leucine --> valine mutants is caused by ImH ligand orientational dynamics. The two angular orientations of the ImH ligand could be determined from the (1)H chemical shifts of the heme methyls, and the rate of interconversion of the two forms could be estimated from the NOESY diagonal and cross peak intensities. K(eq) is 100 or larger and favors an orientation similar to that found for the WT NP2-ImH complex.

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.

Models of the low-spin iron(III) hydroperoxide intermediate of heme oxygenase: magnetic resonance evidence for thermodynamic stabilization of the d(xy) electronic state at ambient temperatures.

The (13)C pulsed ENDOR and NMR study of [meso-(13)C-TPPFe(OCH(3))(OO(t)Bu)](-) performed in this work shows that although the unpaired electron in low-spin ferrihemes containing a ROO(-) ligand resides in a d(pi) orbital at 8 K, the d(xy) electron configuration is favored at physiological temperatures. The variable temperature NMR spectra indicate a dynamic situation in which a heme with a d(pi) electron configuration and planar porphyrinate ring is in equilibrium with a d(xy) electron configuration that has a ruffled porphyrin ring. Because of the similarity in the EPR spectra of the hydroperoxide complexes of heme oxygenase, cytochrome P450, and the model heme complex reported herein, it is possible that these two electron configurations and ring conformations may also exist in equilibrium in the enzymatic systems. The ruffled porphyrinate ring would aid the attack of the terminal oxygen of the hydroperoxide intermediate of heme oxygenase (HO) on the meso-carbon, and the large spin density at the meso-carbons of a d(xy) electron configuration heme suggests the possibility of a radical mechanism for HO. The dynamic equilibrium between the ruffled (d(xy)) and planar (d(pi)) conformers observed in the model complexes also suggests that a flexible heme binding cavity may be an important structural motif for heme oxygenase activity.