Gary J. Gerfen

Thiyl Radicals in Ribonucleotide Reductases

The ribonucleoside triphosphate reductase (RTPR) from Lactobacillus leichmannii catalyzes adenosylcobalamin (AdoCbl)-dependent nucleotide reduction, as well as exchange of the 5′ hydrogens of AdoCbl with solvent. A protein-based thiyl radical is proposed as an intermediate in both of these processes. In the presence of RTPR containing specifically deuterated cysteine residues, the electron paramagnetic resonance (EPR) spectrum of an intermediate in the exchange reaction and the reduction reaction, trapped by rapid freeze quench techniques, exhibits narrowed hyperfine features relative to the corresponding unlabeled RTPR. The spectrum was interpreted to represent a thiyl radical coupled to cob(II)alamin. Another proposed intermediate, 5′-deoxyadenosine, was detected by rapid acid quench techniques. Similarities in mechanism between RTPR and the Escherichia coli ribonucleotide reductase suggest that both enzymes require a thiyl radical for catalysis.

High frequency (140 GHz) dynamic nuclear polarization: Polarization transfer to a solute in frozen aqueous solution

Dynamic nuclear polarization (DNP) transfers the large polarization of unpaired electrons to nuclei and thus significantly enhances the signal strength in nuclear magnetic resonance (NMR) spectroscopy. High frequency/field (140 GHz/5 T) DNP has been implemented in solid state NMR experiments using a nitroxide radical as the paramagnetic polarizing agent in a water:glycerol frozen solution. The 1H and 13C NMR signal strengths of both the solvent and an amino acid solute have been enhanced by a factor of 185, which represents a reduction of ≳102 in sample size requirements or ≳104 in signal acquisition time.

Mechanism of dynamic nuclear polarization in high magnetic fields

Solid-state NMR signal enhancements of about two orders of magnitude (100–400) have been observed in dynamic nuclear polarization (DNP) experiments performed at high magnetic field (5 T) and low temperature (10 K) using the nitroxide radical 4-amino TEMPO as the source of electron polarization. Since the breadth of the 4-amino TEMPO EPR spectrum is large compared to the nuclear Larmor frequency, it has been assumed that thermal mixing (TM) is the dominate mechanism by which polarization is transferred from electron to nuclear spins. However, theoretical explanations of TM generally assume a homogeneously broadened EPR line and, since the 4-amino TEMPO line at 5 T is inhomogeneously broadened, they do not explain the observed DNP enhancements. Accordingly, we have developed a treatment of DNP that explicitly uses electron–electron cross-relaxation to mediate electron–nuclear polarization transfer. The process proceeds via spin flip–flops between pairs of electronic spin packets whose Zeeman temperatures di...

Biosynthesis and Functions of a Melanoid Pigment Produced by Species of the Sporothrix Complex in the Presence of l-Tyrosine

ABSTRACT Sporothrix schenckii is the etiological agent of sporotrichosis, the main subcutaneous mycosis in Latin America. Melanin is an important virulence factor of S. schenckii, which produces dihydroxynaphthalene melanin (DHN-melanin) in conidia and yeast cells. Additionally, l-dihydroxyphenylalanine (l-DOPA) can be used to enhance melanin production on these structures as well as on hyphae. Some fungi are able to synthesize another type of melanoid pigment, called pyomelanin, as a result of tyrosine catabolism. Since there is no information about tyrosine catabolism in Sporothrix spp., we cultured 73 strains, including representatives of newly described Sporothrix species of medical interest, such as S. brasiliensis, S. schenckii, and S. globosa, in minimal medium with tyrosine. All strains but one were able to produce a melanoid pigment with a negative charge in this culture medium after 9 days of incubation. An S. schenckii DHN-melanin mutant strain also produced pigment in the presence of tyrosine. Further analysis showed that pigment production occurs in both the filamentous and yeast phases, and pigment accumulates in supernatants during stationary-phase growth. Notably, sulcotrione inhibits pigment production. Melanin ghosts of wild-type and DHN mutant strains obtained when the fungus was cultured with tyrosine were similar to melanin ghosts yielded in the absence of the precursor, indicating that this melanin does not polymerize on the fungal cell wall. However, pyomelanin-producing fungal cells were more resistant to nitrogen-derived oxidants and to UV light. In conclusion, at least three species of the Sporothrix complex are able to produce pyomelanin in the presence of tyrosine, and this pigment might be involved in virulence.

Observation of organometallic and radical intermediates formed during the reaction of methyl-coenzyme M reductase with bromoethanesulfonate.

Methyl-coenzyme M reductase (MCR) from methanogenic archaea catalyzes the final step of methane formation, in which methyl-coenzyme M (2-methylthioethanesulfonate, methyl-SCoM) is reduced with coenzyme B (N-(7-mercaptoheptanoyl)threonine phosphate, CoBSH) to form methane and the heterodisulfide CoBS-SCoM. The active dimeric form of MCR contains two Ni(I)-F(430) prosthetic groups, one in each monomer. This report describes studies of the reaction of the active Ni(I) state of MCR (MCR(red1)) with BES (2-bromoethanesulfonate) and CoBSH or its analogue, CoB(6)SH (N-(6-mercaptohexanoyl)threonine phosphate), by transient kinetic measurements using EPR and UV-visible spectroscopy and by global fits of the data. This reaction is shown to lead to the formation of three intermediates, the first of which is assigned as an alkyl-Ni(III) species that forms as the active Ni(I)-MCR(red1) state of the enzyme decays. Subsequently, a radical (MCR(BES) radical) is formed that was characterized by multifrequency electron paramagnetic resonance (EPR) studies at X- ( approximately 9 GHz), Q- ( approximately 35 GHz), and D- ( approximately 130 GHz) bands and by electron-nuclear double resonance (ENDOR) spectroscopy. The MCR(BES) radical is characterized by g-values at 2.00340 and 1.99832 and includes a strongly coupled nonexchangeable proton with a hyperfine coupling constant of 50 MHz. Based on transient kinetic measurements, the formation and decay of the radical coincide with a species that exhibits absorption peaks at 426 and 575 nm. Isotopic substitution, multifrequency EPR, and ENDOR spectroscopic experiments rule out the possibility that MCR(BES) is a tyrosyl radical and indicate that if a tyrosyl radical is formed during the reaction, it does not accumulate to detectable levels. The results provide support for a hybrid mechanism of methanogenesis by MCR that includes both alkyl-Ni and radical intermediates.

Electronic structure and hyperfine interactions in thioether-substituted tyrosyl radicals

Abstract Electron-magnetic resonance spectroscopy and computational studies of the cysteine cross-linked tyrosyl radical in apogalactose oxidase have led to conflicting ideas regarding spectral assignments and protein-environment effects. We report DFT calculations on model radicals that clarify these issues. Calculated Fermi contact interactions do not resolve the ambiguity in spectral assignments; better insight is provided by the anisotropy of the hyperfine interactions and the systematic effects of thioether substitution. DFT results on model systems do not account for salient properties of the apogalactose radical. This inadequacy suggests that the protein environment exerts significant effects on the electronic structure of the radical.

Structural comparisons of arachidonic acid-induced radicals formed by prostaglandin H synthase-1 and -2

Cyclooxygenase catalysis by prostaglandin H synthase (PGHS)-1 and -2 involves reaction of a peroxide-induced Tyr385 radical with arachidonic acid (AA) to form an AA radical that reacts with O(2). The potential for isomeric AA radicals and formation of an alternate tyrosyl radical at Tyr504 complicate analysis of radical intermediates. We compared the EPR spectra of PGHS-1 and -2 reacted with peroxide and AA or specifically deuterated AA in anaerobic, single-turnover experiments. With peroxide-treated PGHS-2, the carbon-centered radical observed after AA addition was consistently a pentadienyl radical; a variable wide-singlet (WS) contribution from mixture of Tyr385 and Tyr504 radicals was also present. Analogous reactions with PGHS-1 produced EPR signals consistent with varying proportions of pentadienyl and tyrosyl radicals, and two additional EPR signals. One, insensitive to oxygen exposure, is the narrow singlet tyrosyl radical with clear hyperfine features found previously in inhibitor-pretreated PGHS-1. The second type of EPR signal is a narrow singlet lacking detailed hyperfine features that disappeared upon oxygen exposure. This signal was previously ascribed to an allyl radical, but high field EPR analysis indicated that ~90% of the signal originates from a novel tyrosyl radical, with a small contribution from a carbon-centered species. The radical kinetics could be resolved by global analysis of EPR spectra of samples trapped at various times during anaerobic reaction of PGHS-1 with a mixture of peroxide and AA. The improved understanding of the dynamics of AA and tyrosyl radicals in PGHS-1 and -2 will be useful for elucidating details of the cyclooxygenase mechanism, particularly the H-transfer between tyrosyl radical and AA.