Tatjana Kh. Shokhireva

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 1H 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. Keq is 100 or larger and favors an orientation similar to that found for the WT NP2–ImH complex.

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

Chloroiron meso-triphenylcorrolates: electronic ground state and spin delocalization

Abstract Four chloroiron meso -triphenyl-substituted corrolates have been synthesized and studied by 1 H NMR spectroscopy. As in the case of the β-pyrrole-octaalkylcorrolatoiron chloride complexes studied previously [Inorg. Chem. 39 (2000) 3466], these complexes were also found to be S=3/2 Fe(III) corrolate( 2− ) π-cation radical species, where the macrocycle radical electron is antiferromagnetically coupled to the metal electrons to give an overall S=1 complex. This conclusion is based upon the large alternating-sign contact shifts observed for the meso -phenyl protons. The 1 H isotropic shifts of the pyrrole-H of these chloroiron–triphenylcorrolate complexes are similar to those of the chloroiron tri-(pentafluorophenyl)corrolate complex reported previously and said to be a S=1 Fe(IV) complex bound to a simple corrolate( 3− ) ligand [Inorg. Chem. 39 (2000) 2704]. The 19 F NMR spectrum of the latter complex shows that it has small (negative) phenyl-F isotropic shifts for all phenyl-F, which might suggest that this single compound has a different electronic structure than all other chloroiron corrolates investigated thus far. However, there have as yet been very few NMR investigations of paramagnetic metal macrocycles having fluorine substituents, and thus it is premature to conclude that the small phenyl-F isotropic shifts are definitive proof of small spin density at the meso positions of the corrolate ring. It is concluded that pyrrole-H chemical shifts alone cannot differentiate the two possible electron configurations, simple S=1 Fe(IV) (Corr 3− ) and antiferromagnetically coupled S=3/2 Fe(III) (Corr 2− ), and that based on the 1 H investigations reported in this and two previous papers, all chloroiron corrolates reported thus far, with the exception of one, have the electron configuration S=3/2 Fe(III) (Corr 2− ), in which the corrolate unpaired electron is antiferromagnetically coupled to the three metal electrons, yielding an overall spin for the complex, S=1. The electron configuration of the one exception, the strongly electron-withdrawing tri-(pentafluorophenyl)corrolate complex of iron chloride, cannot as yet be definitively assigned.

Rates of axial ligand rotation in diamagnetic d6 Co(III) and Fe(II) porphyrinates

Abstract In order to investigate the rates of rotation of pyridine and imidazole ligands in diamagnetic low-spin d 6 Co(III) and Fe(II) porphyrinate systems, we have synthesized tetramesitylporphyrinate (TMP) complexes of each of these metals with pyridine and imidazole ligands and investigated them as a function of temperature by 1 H NMR spectroscopy. We have already reported that for TMPFe(III) and -Co(III) complexes with hindered imidazoles the TMP o -CH 3 resonances can be used to measure the rates of rotation (N.V. Shokhirev, T.Kh. Shokhireva, J.R. Polam, C.T. Watson, K. Raffi, U. Simonis and F.A. Walker, J. Phys. Chem. A, 101 (1997) 2778). For the bis-1,2-dimethylimidazole complex, [TMPCo(1,2-Me 2 Im) 2 ]BF 4 , at ambient temperatures ligand rotation is slow but measureable on the NMP time scale, and four o -CH 3 resonances are observed, as we have already reported. In contrast, as shown in the present work, for the bis-4-dimethylaminopyridine complex, [TMPCo(4-NMe 2 Py) 2 ]BF 4 , ligand rotation is extremely rapid at ambient temperatures. At temperatures below −50°C at 300 MHz the o -CH 3 resonance broadens and the rates of rotation can be estimated using the modified Bloch equations simplified for the fast exchange regime. The activation parameters ΔH ≠ and ΔS ≠ have been determined, and the extrapolated rate constant at 25°C, k ex ≥ 1.1 × 10 6 s −1 . These results contradict previous reports (J. Huet and A. Gaudemer, Org. Magn. Reson., 15 (1981) 347; I. Cassidei, H. Bang, J.O. Edwards and R. G. Lawler, J. Phys. Chem., 95 (1991) 7186) that pyridine ligands bound to Co(III) porphyrinates do not rotate at room temperature in homogeneous solution. For unhindered imidazole complexes, such as [TMPCo(NMeIm) 2 ] + BF 4 − , no broadening of the o -CH 3 resonance is observed, even at −90°C, and thus the rate of axial ligand rotation is too fast to measure, even at that low temperature (or the difference in chemical shift of the two resonances expected if ligand rotation is slow is very small). For the corresponding Fe(II) porphyrinate complexes, the rates of pyridine and unhindered imidazole rotation are too fast to measure, even at −90°C. The 2-methylimidazole complex undergoes chemical reactions that prevent detailed study of this system by NMR spectroscopy, but the 1,2-dimethylimidazole complex is stable and of similar structure (ruffled porphyrinate ring, axial ligands in perpendicular planes) to the Co(III) and Fe(III) analogs, with the rate constant for ligand rotation, k ex ∼ 1 s −1 , at −90°C. Assuming a similar activation enthalpy to those of the Co(III) and Fe(III) systems, the rate of rotation of axial ligands in [TMPFe(1,2-Me 2 Im) 2 ] at 25°C is estimated to be about 2 × 10 4 s −1 .

ROESY spectra of paramagnetic model heme complexes: unambiguous differentiation of cross-peaks due to chemical exchange and the nuclear overhauser effect in the same molecule

Abstract We have investigated the ROESY spectrum of [TMPFe(2-MelmH) 2 ] ClO 4 over the temperature range from −55 tc −75°C using spinlock fields of 4.1 and 10.3 kHz. The latter B 1 field is more typical of those used to observe TOCSY spectra, but is more closely in line with the spectral bandwidth required for this paramagnetic complex (12 kHz at 300 MHz). Chemical exchange cross-peaks were easily observed at both spin-lock fields, but ROE cross-peaks were significantly more intense with the larger B 1 field. At −75°C, all but one of the expected ROEs were observed; the absent ROE is that between the 2-CH 3 of the axial ligands and either the ortho -CH 3 (2) or −(3) resonance. The absence of this ROE is probably due to the short T 1 of the ligand 2-CH 1 protons ( T 1 between it and the ortho -CH 1 protons (25–35 ms at −75°C). In spite of the absence of this ROE a nearly complete assignment of the ROESY spectrum in terms of the structure of the complex has been made. Minor interference from TOCSY effects, observed as a decrease in the expected intensity of the ROE cross-peaks between pyrrole-H resonances (2) and (4), was noted, but did not prevent observation of this ROE. For the diamagnetic Co(III) analog, both this ROE (to ortho -CH 1 (3) in this case) and the ROE between the ligand 4-H and ortho -CH 3 (4) resonances were observed, but the small separation between pyrrole-H signals precludes a complete assignment of the ROESY spectrum of this complex.