Muralidharan Shanmugam

Magnetic Anisotropy of Dysprosium(III) in a Low-Symmetry Environment: A Theoretical and Experimental Investigation

A mixed theoretical and experimental approach was used to determine the local magnetic anisotropy of the dysprosium(III) ion in a low-symmetry environment. The susceptibility tensor of the monomeric species having the formula [Dy(hfac)(3)(NIT-C(6)H(4)-OEt)(2)], which contains nitronyl nitroxide (NIT-R) radicals, was determined at various temperatures through angle-resolved magnetometry. These results are in agreement with ab initio calculations performed using the complete active space self-consistent field (CASSCF) method, validating the predictive power of this theoretical approach for complex systems containing rare-earth ions, even in low-symmetry environments. Susceptibility measurements performed with the applied field along the easy axis eventually permitted a detailed analysis of the temperature and field dependence of the magnetization, providing evidence that the Dy ion transmits an antiferromagnetic interaction between radicals but that the Dy-radical interaction is ferromagnetic.

Metal cages using a bulky phosphonate as a ligand

The synthesis, structure, magnetic and electronic properties of soluble transition metal phosphonate cages utilizing tritylphosphonic acid (TPA) as ligand are reported.

Probing the oxyferrous and catalytically active ferryl states of Amphitrite ornata dehaloperoxidase by cryoreduction and EPR/ENDOR spectroscopy. Detection of compound I.

Dehaloperoxidase (DHP) from Amphitrite ornata is a heme protein that can function both as a hemoglobin and as a peroxidase. This report describes the use of 77 K cryoreduction EPR/ENDOR techniques to study both functions of DHP. Cryoreduced oxyferrous [Fe(II)-O(2)] DHP exhibits two EPR signals characteristic of a peroxoferric [Fe(III)-O(2)(2-)] heme species, reflecting the presence of conformational substates in the oxyferrous precursor. (1)H ENDOR spectroscopy of the cryogenerated substates shows that H-bonding interactions between His N(ε)H and heme-bound O(2) in these conformers are similar to those in the β-chain of oxyferrous hemoglobin A (HbA) and oxyferrous myoglobin, respectively. Decay of cryogenerated peroxoferric heme DHP intermediates upon annealing at temperatures above 180 K is accompanied by the appearance of a new paramagnetic species with an axial EPR signal with g(⊥) = 3.75 and g(∥) = 1.96, characteristic of an S = 3/2 spin state. This species is assigned to Compound I (Cpd I), in which a porphyrin π-cation radical is ferromagnetically coupled with an S = 1 ferryl [Fe(IV)═O] ion. This species was also trapped by rapid freeze-quench of the ambient-temperature reaction mixture of ferric [Fe(III)] DHP and H(2)O(2). However, in the latter case Cpd I is reduced very rapidly by a nearby tyrosine to form Cpd ES [(Fe(IV)═O)(porphyrin)/Tyr(•)]. Addition of the substrate analogue 2,4,6-trifluorophenol (F(3)PhOH) suppresses formation of the Cpd I intermediate during annealing of cryoreduced oxyferrous DHP at 190 K but has no effect on the spectroscopic properties of the remaining cryoreduced oxyferrous DHP intermediates and kinetics of their decay. These observations indicate that substrate (i) binds to oxyferrous DHP outside of the distal pocket and (ii) can reduce Cpd I to Cpd II [Fe(IV)═O]. These assumptions are also supported by the observation that F(3)PhOH has only a small effect on the EPR properties of radiolytically cryooxidized and cryoreduced ferrous [Fe(II)] DHP. EPR spectra of cryoreduced ferrous DHP disclose the multiconformational nature of the ferrous DHP precursor. The observation and characterization of Cpds I, II, and ES in the absence and in the presence of F(3)PhOH provides definitive evidence of a mechanism involving consecutive one-electron steps and clarifies the role of all intermediates formed during turnover.

Minor changes in phosphonate ligands lead to new hexa- and dodeca-nuclear Mn clusters

Drastic changes in the structure of manganese clusters are observed from a minor modification of a phosphonate ligand.

Identification of protonated oxygenic ligands of ribonucleotide reductase intermediate X.

We previously used a combination of continuous-wave (CW) and pulsed electron-nuclear double resonance (ENDOR) protocols to identify the types of protonated oxygen (OH(x)) species and their disposition within the Fe(III)/Fe(IV) cluster of intermediate X, the direct precursor of the essential diferric-tyrosyl radical cofactor of the beta2 subunit of Escherichia coli ribonucleotide reductase (RNR). We concluded that X contains the [(H(x)O)Fe(III)OFe(IV)] fragment (T model), and does not contain a mu-hydroxo bridge. When combined with a subsequent (17)O ENDOR study of X prepared with H(2)(17)O and with (17)O(2), the results led us to suggest that this fragment is the entire inorganic core of X. This has been questioned by recent reports, but these reports do not themselves agree on the core of X. One concluded that X possesses a di-mu-oxo Fe(III)/Fe(IV) core plus a terminal (H(2)O) bound to Fe(III) [e.g., Han, W.-G.; Liu, T.; Lovell, T.; Noodleman, L. J. Am. Chem. Soc. 2005, 127, 15778-15790]. The other [Mitic, N.; Clay, M. D.; Saleh, L.; Bollinger, J. M.; Solomon, E. I. J. Am. Chem. Soc. 2007, 129, 9049-9065] concluded that X contains only a single oxo bridge and postulated the presence of an additional hydroxo bridge plus a terminal hydroxyl bound to Fe(III). In this report we take advantage of improvements in 35 GHz pulsed ENDOR performance to reexamine the protonation state of oxygenic ligands of the inorganic core of X by directly probing the exchangeable proton(s) with (2)H pulsed ENDOR spectroscopy. These (2)H ENDOR measurements confirm that X contains an Fe(III)-bound terminal aqua ligand (H(x)O), but the spectra contain none of the features that would be required for the proton of a bridging hydroxyl. Thus, we confirm that X contains a terminal aqua (most likely hydroxo) ligand to Fe(III) in addition to one or two mu-oxo bridges but does not contain a mu-hydroxo bridge. The (2)H ENDOR measurements further demonstrate that this conclusion is applicable to both wild type and Y122F-beta2 mutant, and in fact we detect no difference between the properties of protons on the terminal oxygens in the two variants; likewise, (14)N ENDOR measurements of histidyl ligands bound to Fe show no difference between the two variants.

13C and 63,65Cu ENDOR studies of CO Dehydrogenase from Oligotropha carboxidovorans. Experimental Evidence in Support of a Copper–Carbonyl Intermediate

We report here an ENDOR study of an S = 1/2 intermediate state trapped during reduction of the binuclear Mo/Cu enzyme CO dehydrogenase by CO. ENDOR spectra of this state confirm that the (63,65)Cu nuclei exhibits strong and almost entirely isotropic coupling to the unpaired electron, show that this coupling atypically has a positive sign, aiso = +148 MHz, and indicate an apparently undetectably small quadrupolar coupling. When the intermediate is generated using (13)CO, coupling to the (13)C is observed, with aiso = +17.3 MHz. A comparison with the couplings seen in related, structurally assigned Mo(V) species from xanthine oxidase, in conjunction with complementary computational studies, leads us to conclude that the intermediate contains a partially reduced Mo(V)/Cu(I) center with CO bound at the copper. Our results provide strong experimental support for a reaction mechanism that proceeds from a comparable complex of CO with fully oxidized Mo(VI)/Cu(I) enzyme.

Role of the Proximal Cysteine Hydrogen Bonding Interaction in Cytochrome P450 2B4 Studied by Cryoreduction, Electron Paramagnetic Resonance, and Electron-Nuclear Double Resonance Spectroscopy.

Crystallographic studies have shown that the F429H mutation of cytochrome P450 2B4 introduces an H-bond between His429 and the proximal thiolate ligand, Cys436, without altering the protein fold but sharply decreases the enzymatic activity and stabilizes the oxyferrous P450 2B4 complex. To characterize the influence of this hydrogen bond on the states of the catalytic cycle, we have used radiolytic cryoreduction combined with electron paramagnetic resonance (EPR) and (electron-nuclear double resonance (ENDOR) spectroscopy to study and compare their characteristics for wild-type (WT) P450 2B4 and the F429H mutant. (i) The addition of an H-bond to the axial Cys436 thiolate significantly changes the EPR signals of both low-spin and high-spin heme-iron(III) and the hyperfine couplings of the heme-pyrrole (14)N but has relatively little effect on the (1)H ENDOR spectra of the water ligand in the six-coordinate low-spin ferriheme state. These changes indicate that the H-bond introduced between His and the proximal cysteine decreases the extent of S → Fe electron donation and weakens the Fe(III)-S bond. (ii) The added H-bond changes the primary product of cryoreduction of the Fe(II) enzyme, which is trapped in the conformation of the parent Fe(II) state. In the wild-type enzyme, the added electron localizes on the porphyrin, generating an S = (3)/2 state with the anion radical exchange-coupled to the Fe(II). In the mutant, it localizes on the iron, generating an S = (1)/2 Fe(I) state. (iii) The additional H-bond has little effect on g values and (1)H-(14)N hyperfine couplings of the cryogenerated, ferric hydroperoxo intermediate but noticeably slows its decay during cryoannealing. (iv) In both the WT and the mutant enzyme, this decay shows a significant solvent kinetic isotope effect, indicating that the decay reflects a proton-assisted conversion to Compound I (Cpd I). (v) We confirm that Cpd I formed during the annealing of the cryogenerated hydroperoxy intermediate and that it is the active hydroxylating species in both WT P450 2B4 and the F429H mutant. (vi) Our data also indicate that the added H-bond of the mutation diminishes the reactivity of Cpd I.

Carbene in Cupredoxin Protein Scaffolds: Replacement of a Histidine Ligand in the Active Site Substantially Alters Copper Redox Properties

N-heterocyclic carbene (NHC) ligands have had a major impact in homogeneous catalysis, however, their potential role in biological systems is essentially unexplored. We replaced a copper-coordinating histidine (His) in the active site of the redox enzyme azurin with exogenous dimethyl imidazolylidene. This NHC rapidly restores the type-1 Cu center, with spectroscopic properties (EPR, UV/Vis) that are identical to those from N-coordination of the His in the wild type. However, the introduction of the NHC markedly alters the redox potential of the metal, which is a key functionality of this blue copper protein. These results suggest that C-bonding for histidine is plausible and a potentially relevant bonding mode of redox-active metalloenzymes in their (transient) active states.