Matthew D. Krzyaniak

Hyperfine interactions of narrow-line trityl radical with solvent molecules.

The electron nuclear dipolar interactions responsible for some dynamic nuclear polarization (DNP) mechanisms also are responsible for the presence formally in CW EPR spectra of forbidden satellite lines in which both the electron spin and a nuclear spin flip. Such lines arising from (1)H nuclei are easily resolved in CW EPR measurements of trityl radicals, a popular family of DNP reagents. The satellite lines overlap some of the hyperfine features from (13)C in natural abundance in the trityl radical, but their intensity can be easily determined by simple simulations of the EPR spectra using the hyperfine parameters of the trityl radical. Isotopic substitution of (2)H for (1)H among the hydrogens of the trityl radical and/or the solvent allows the dipolar interactions from the (1)H on the trityl radical and from the solvent to be determined. The intensity of the dipolar interactions, integrated over all the (1)H in the system, is characterized by the traditional parameter called reff. For the so-called Finland trityl in methanol, the reff values indicate that collectively the (1)H in the unlabeled solvent have a stronger integrated dipolar interaction with the unpaired electron spin of the Finland trityl than do the (1)H in the radical and consequently will be a more important DNP route. Although reff has the dimensions of distance, it does not correspond to any simple physical dimension in the trityl radical because the details of the unpaired electron spin distribution and the hydrogen distribution are important in the case of trityls.

EPR evidence of cyanide binding to the Mn(Mg) center of cytochrome c oxidase: support for Cu(A)-Mg involvement in proton pumping.

We examined the anion binding behavior of the Mg(Mn) site in cytochrome c oxidase to test a possible role of this center in proton pumping. Rhodobacter sphaeroides grown in a Mn(II)-rich medium replaces the intrinsic Mg(II) ion with an EPR-detectable Mn(II) ion without change in activity. Due to its close proximity and a shared ligand, oxidized Cu(A) is spin-coupled to the Mn(II) ion, affecting the EPR spectrum. An examination of both bovine and R.s. oxidase crystal structures reveals a hydrogen-bonding pattern in the vicinity of the Mg(II) site that is consistent with three water ligands of the Mg(Mn) center when Cu(A) is oxidized. In the reduced structure, one water molecule in the vicinity of the Cu(A) ligand, E198, moves closer, appearing to be converted into an ionically bonded hydronium ion, while a second water molecule bonded to Mg(Mn) shows evidence of conversion to a hydroxide. The implied proton movement is proposed to be part of a redox-linked export of a pumped proton from the binuclear center into the exit pathway. To test the model, cyanide and azide were added to the oxidized and reduced forms of the enzyme, and Mn(II) CW-EPR and ESEEM spectra were recorded. Addition of azide broadened the CW-EPR spectra for both oxidized and reduced enzyme. Cyanide addition affected the Mn(II) CW-EPR spectrum of reduced cytochrome c oxidase by increasing Mn(II) zero field splitting and broadening the spectral line shapes but had no effect on oxidized enzyme. ESEEM measurements support a differential ability of Mn(II) to bind cyanide in the reduced state of cytochrome c oxidase. This new observation of anion binding at the Mg/Mn site is of interest in terms of accessibility of the buried site and its potential role in redox-dependent proton pumping.

Strength of axial water ligation in substrate-free cytochrome P450s is isoform dependent

The heme-containing cytochrome P450s exhibit isoform-dependent ferric spin equilibria in the resting state and differential substrate-dependent spin equilibria. The basis for these differences is not well understood. Here, magnetic circular dichroism (MCD) reveals significant differences in the resting low spin ligand field of CYPs 3A4, 2E1, 2C9, 125A1, and 51B1, which indicates differences in the strength of axial water ligation to the heme. The near-infrared bands that specifically correspond to charge-transfer porphyrin-to-metal transitions span a range of energies of nearly 2 kcal/mol. In addition, the experimentally determined MCD bands are not entirely in agreement with the expected MCD energies calculated from electron paramagnetic resonance parameters, thus emphasizing the need for the experimental data. MCD marker bands of the high spin heme between 500 and 680 nm were also measured and suggest only a narrow range of energies for this ensemble of high spin Cys(S–) → Fe3+ transitions among these isoforms. The differences in axial ligand energies between CYP isoforms of the low spin states likely contribute to the energetics of substrate-dependent spin state perturbation. However, these ligand field energies do not correlate with the fraction of high spin vs low spin in the resting state enzyme, suggestive of differences in water access to the heme or isoform-dependent differences in the substrate-free high spin states as well.

A Caged, Destabilized, Free Radical Intermediate in the Q‐Cycle

The Rieske/cytochrome b complexes, also known as cytochrome bc complexes, catalyze a unique oxidant‐induced reduction reaction at their quinol oxidase (Qo) sites, in which substrate hydroquinone reduces two distinct electron transfer chains, one through a series of high‐potential electron carriers, the second through low‐potential cytochrome b. This reaction is a critical step in energy storage by the Q‐cycle. The semiquinone intermediate in this reaction can reduce O2 to produce deleterious superoxide. It is yet unknown how the enzyme controls this reaction, though numerous models have been proposed. In previous work, we trapped a Q‐cycle semiquinone anion intermediate, termed SQo, in bacterial cytochrome bc1 by rapid freeze‐quenching. In this work, we apply pulsed‐EPR techniques to determine the location and properties of SQo in the mitochondrial complex. In contrast to semiquinone intermediates in other enzymes, SQo is not thermodynamically stabilized, and can even be destabilized with respect to solution. It is trapped in Qo at a site that is distinct from previously described inhibitor‐binding sites, yet sufficiently close to cytochrome bL to allow rapid electron transfer. The binding site and EPR analyses show that SQo is not stabilized by hydrogen bonds to proteins. The formation of SQo involves “stripping” of both substrate ‐OH protons during the initial oxidation step, as well as conformational changes of the semiquinone and Qo proteins. The resulting charged radical is kinetically trapped, rather than thermodynamically stabilized (as in most enzymatic semiquinone species), conserving redox energy to drive electron transfer to cytochrome bL while minimizing certain Q‐cycle bypass reactions, including oxidation of prereduced cytochrome b and reduction of O2.

Pulsed EPR study of amino acid and tetrahydropterin binding in a tyrosine hydroxylase nitric oxide complex: evidence for substrate rearrangements in the formation of the oxygen-reactive complex.

Tyrosine hydroxylase is a nonheme iron enzyme found in the nervous system that catalyzes the hydroxylation of tyrosine to form l-3,4-dihydroxyphenylalanine, the rate-limiting step in the biosynthesis of the catecholamine neurotransmitters. Catalysis requires the binding of three substrates: tyrosine, tetrahydrobiopterin, and molecular oxygen. We have used nitric oxide as an O₂ surrogate to poise Fe(II) at the catalytic site in an S = 3/2, {FeNO}⁷ form amenable to EPR spectroscopy. ²H-electron spin echo envelope modulation was then used to measure the distance and orientation of specifically deuterated substrate tyrosine and cofactor 6-methyltetrahydropterin with respect to the magnetic axes of the {FeNO}⁷ paramagnetic center. Our results show that the addition of tyrosine triggers a conformational change in the enzyme that reduces the distance from the {FeNO}⁷ center to the closest deuteron on 6,7-²H-6-methyltetrahydropterin from >5.9 Å to 4.4 ± 0.2 Å. Conversely, the addition of 6-methyltetrahydropterin to enzyme samples treated with 3,5-²H-tyrosine resulted in reorientation of the magnetic axes of the S = 3/2, {FeNO}⁷ center with respect to the deuterated substrate. Taken together, these results show that the coordination of both substrate and cofactor direct the coordination of NO to Fe(II) at the active site. Parallel studies of a quaternary complex of an uncoupled tyrosine hydroxylase variant, E332A, show no change in the hyperfine coupling to substrate tyrosine and cofactor 6-methyltetrahydropterin. Our results are discussed in the context of previous spectroscopic and X-ray crystallographic studies done on tyrosine hydroxylase and phenylalanine hydroxylase.

Drug modulation of water-heme interactions in low-spin P450 complexes of CYP2C9d and CYP125A1.

Azoles and pyridines are commonly incorporated into small molecule inhibitor scaffolds that target cytochromes P450 (CYPs) as a strategy to increase drug binding affinity, impart isoform-dependent selectivity, and improve metabolic stability. Optical absorbance spectra of the CYP-inhibitor complex are widely used to infer whether these inhibitors are ligated directly to the heme iron as catalytically inert, low-spin (type II) complexes. Here, we show that the low-spin complex between a drug-metabolizing CYP2C9 variant and 4-(3-phenylpropyl)-1H-1,2,3-triazole (PPT) retains an axial water ligand despite exhibiting elements of classic type II optical behavior. Hydrogens of the axial water ligand are observed by pulsed electron paramagnetic resonance (EPR) spectroscopy for both inhibitor-free and inhibitor-bound species and show that inhibitor binding does not displace the axial water. A (15)N label incorporated into PPT is 0.444 nm from the heme iron, showing that PPT is also in the active site. The reverse type I inhibitor, LP10, of CYP125A1 from Mycobacterium tuberculosis, known from X-ray crystal structures to form a low-spin water-bridged complex, is found by EPR and by visible and near-infrared magnetic circular dichroism spectroscopy to retain the axial water ligand in the complex in solution.

EPR study of the astaxanthin n-octanoic acid monoester and diester radicals on silica-alumina.

The radical intermediates of the n-octanoic monoester and n-octanoic diester of astaxanthin were detected by pulsed EPR measurements carried out on the UV-produced radicals on silica-alumina artificial matrix and characterized by density functional theory (DFT) calculations. Previous Mims ENDOR for astaxanthin detected the radical cation and neutral radicals formed by proton loss from the C3 (or C3) position and from the methyl groups. Deprotonation of the astaxanthin neutral radical formed at the C3 (or C3) position resulted in a radical anion. DFT calculations for astaxanthin showed that the lowest energy neutral radical forms by proton loss at the C3 (or C3) position of the terminal ring followed by proton loss at the methyl groups of the polyene chain. Contrary to astaxanthin where proton loss can occur at either end of the symmetrical radical, for the diester of astaxanthin, this loss is prevented at the cyclohexene ends and is favored for its methyl groups. The monoester of astaxanthin, however, allows formation of the neutral radical at C3 and prevents its formation at the opposite end where the ester group is attached. At the terminal ring without the ester group attached, migration of proton from hydroxyl group to carbonyl group facilitates resonance stabilization, similarly to already published results for astaxanthin. However, cw EPR shows no evidence of a monoester radical anion formed. This study suggests the different radicals of astaxanthin and its esters that would form in a preferred environment, either hydrophobic or hydrophilic, depending on their structure.

DFT and ENDOR Study of Bixin Radical Cations and Neutral Radicals on Silica–Alumina

Bixin, a carotenoid found in annatto (Bixa orellana), is unique among natural carotenoids by being water-soluble. We stabilized free radicals from bixin on the surface of silica-alumina (Si-Al) and characterized them by pulsed electron-nuclear double resonance (ENDOR). DFT calculations of unpaired electron spin distribution for various bixin radicals predict the EPR hyperfine couplings. Least-square fitting of experimental ENDOR spectra by spectra calculated from DFT hyperfine couplings characterized the radicals trapped on Si-Al. DFT predicts that the trans bixin radical cation is more stable than the cis bixin radical cation by 1.26 kcal/mol. This small energy difference is consistent with the 26% trans and 23% cis radical cations in the ENDOR spectrum. The remainder of the ENDOR spectrum is due to several neutral radicals formed by loss of a H(+) ion from the 9, 9, 13, or 13 methyl group, a common occurrence in all water-insoluble carotenoids previously studied. Although carboxyl groups of bixin strongly affect its solubility relative to other natural carotenoids, they do not alter properties of its free radicals based on DFT calculations and EPR measurements which remain similar to typical water-insoluble carotenoids.

HYSCORE Analysis of the Effects of Substrates on Coordination of Water to the Active Site Iron in Tyrosine Hydroxylase

Tyrosine hydroxylase is a mononuclear non-heme iron monooxygenase found in the central nervous system that catalyzes the hydroxylation of tyrosine to yield L-3,4-dihydroxyphenylalanine, the rate-limiting step in the biosynthesis of catecholamine neurotransmitters. Catalysis requires the binding of tyrosine, a tetrahydropterin, and O₂ at an active site that consists of a ferrous ion coordinated facially by the side chains of two histidines and a glutamate. We used nitric oxide as a surrogate for O₂ to poise the active site iron in an S = ³/₂ {FeNO}⁷ form that is amenable to electron paramagnetic resonance (EPR) spectroscopy. The pulsed EPR method of hyperfine sublevel correlation (HYSCORE) spectroscopy was then used to probe the ligands at the remaining labile coordination sites on iron. For the complex formed by the addition of tyrosine and nitric oxide, TyrH/NO/Tyr, orientation-selective HYSCORE studies provided evidence of the coordination of one H₂O molecule characterized by proton isotropic hyperfine couplings (A(iso) = 0.0 ± 0.3 MHz) and dipolar couplings (T = 4.4 and 4.5 ± 0.2 MHz). These data show complex HYSCORE cross peak contours that required the addition of a third coupled proton, characterized by an A(iso) of 2.0 MHz and a T of 3.8 MHz, to the analysis. This proton hyperfine coupling differed from those measured previously for H₂O bound to {FeNO}⁷ model complexes and was assigned to a hydroxide ligand. For the complex formed by the addition of tyrosine, 6-methyltetrahydropterin, and NO, TyrH/NO/Tyr/6-MPH₄, the HYSCORE cross peaks attributed to H₂O and OH⁻ for the TyrH/NO/Tyr complex were replaced by a cross peak due to a single proton characterized by an A(iso) of 0.0 MHz and a dipolar coupling (T = 3.8 MHz). This interaction was assigned to the N₅ proton of the reduced pterin.

Characterization of Water Coordination to Ferrous Nitrosyl Complexes with fac-N2O, cis-N2O2, and N2O3 Donor Ligands

Electron paramagnetic resonance (EPR) experiments were done on a series of S = (3)/2 ferrous nitrosyl model complexes prepared with chelating ligands that mimic the 2-His-1-carboxylate facial triad iron binding motif of the mononuclear nonheme iron oxidases. These complexes formed a comparative family, {FeNO}(7)(N2Ox)(H2O)3-x with x = 1-3, where the labile coordination sites for the binding of NO and solvent water were fac for x = 1 and cis for x = 2. The continuous-wave EPR spectra of these three complexes were typical of high-spin S = (3)/2 transition-metal ions with resonances near g = 4 and 2. Orientation-selective hyperfine sublevel correlation (HYSCORE) spectra revealed cross peaks arising from the protons of coordinated water in a clean spectral window from g = 3.0 to 2.3. These cross peaks were absent for the {FeNO}(7)(N2O3) complex. HYSCORE spectra were analyzed using a straightforward model for defining the spin Hamiltonian parameters of bound water and showed that, for the {FeNO}(7)(N2O2)(H2O) complex, a single water conformer with an isotropic hyperfine coupling, Aiso = 0.0 ± 0.3 MHz, and a dipolar coupling of T = 4.8 ± 0.2 MHz could account for the data. For the {FeNO}(7)(N2O)(H2O)2 complex, the HYSCORE cross peaks assigned to coordinated water showed more frequency dispersion and were analyzed with discrete orientations and hyperfine couplings for the two water molecules that accounted for the observed orientation-selective contour shapes. The use of three-pulse electron spin echo envelope modulation (ESEEM) data to quantify the number of water ligands coordinated to the {FeNO}(7) centers was explored. For this aspect of the study, HYSCORE spectra were important for defining a spectral window where empirical integration of ESEEM spectra would be the most accurate.