The pulsed electron paramagnetic resonance (EPR) methods of electron spin echo envelope modulation (ESEEM) and electron spin echo-electron nuclear double resonance (ESE-ENDOR) are used to investigate the structure of the Photosystem II oxygen-evolving complex (OEC), including the paramagnetic manganese cluster and its immediate surroundings. Recent unpublished results from the pulsed EPR laboratory at UC-Davis are discussed, along with aspects of recent publications, with a focus on substrate and cofactor interactions. New data on the proximity of exchangeable deuterons around the Mn cluster poised in the S(0)-state are presented and interpreted. These pulsed EPR results are used in an evaluation of several recently proposed mechanisms for PSII water oxidation. We strongly favor mechanistic models where the substrate waters bind within the OEC early in the S-state cycle. Models in which the O-O bond is formed by a nucleophilic attack by a Ca(2+)-bound water on a strong S(4)-state electrophile provide a good match to the pulsed EPR data.

Laccase is a multicopper oxidase that contains four Cu ions, one type 1 (T1), one type 2 (T2), and a coupled binuclear type 3 Cu pair (T3). The T2 and T3 centers form a trinuclear Cu cluster that is the active site for O2 reduction to H2O. A combination of spectroscopic and DFT studies on a derivative where the T1 Cu has been replaced by a spectroscopically innocent Hg2+ ion has led to a detailed geometric and electronic structure description of the resting trinuclear Cu cluster, complementing crystallographic results. The nature of the T2 Cu ligation has been elucidated; this site is three-coordinate with two histidines and a hydroxide over its functional pH range (stabilized by a large inductive effect, cluster charge, and a hydrogen-bonding network). Both the T2 and T3 Cu centers have open coordination positions oriented toward the center of the cluster. DFT calculations show that the negative protein pocket (four conserved Asp/Glu residues within 12 A) and the dielectric of the protein play important roles in the electrostatic stability and integrity of the highly charged, coordinatively unsaturated trinuclear cupric cluster. These tune the ligand binding properties of the cluster, leading to its high affinity for fluoride and its coordination unsaturation in aqueous media, which play a key role in its O2 reactivity.

Background Assay of T cell receptor excision circles (TRECs) in dried blood spots (DBS) obtained at birth permits population-based newborn screening (NBS) for severe combined immunodeficiency (SCID). Objective To report the first 2 years of TREC NBS in California. Methods Since August 2010, California has conducted SCID newborn screening. A high-throughput TREC qPCR assay using DNA isolated from routine DBS was developed. Samples with initial low TREC values had repeat DNA isolation with qPCR for TRECs and a genomic control, and immunophenotyping was performed within the screening program for infants with incomplete or abnormal results. Outcomes were tracked. Results Of 993,724 infants screened, 50 (1/19,900; 0.005%) had significant T cell lymphopenia. Fifteen (1/66,250) required hematopoietic cell or thymus transplantation or gene therapy; these infants had typical SCID (11), leaky SCID or Omenn syndrome (3), or complete DiGeorge syndrome (1). Survival to date in this group is 93%. Other T lymphopenic infants had variant SCID or combined immunodeficiency (6), genetic syndromes associated with T cell impairment (12), secondary T lymphopenia (9) or preterm birth (8). All T lymphopenic infants avoided live vaccines and received appropriate interventions to prevent infections. TREC test specificity was excellent: only 0.08% of infants required a second test and 0.016% required lymphocyte phenotyping by flow cytometry. Conclusions TREC NBS in California has achieved early diagnosis of SCID and other conditions with T lymphopenia, facilitating management and optimizing outcomes. Furthermore, NBS has revealed the incidence, causes and follow-up of T lymphopenia in a large, diverse population.

OBJECTIVES: Newborn screening for severe combined immunodeficiency (SCID) was instituted in California in 2010. In the ensuing 6.5 years, 3 252 156 infants in the state had DNA from dried blood spots assayed for T-cell receptor excision circles (TRECs). Abnormal TREC results were followed-up with liquid blood testing for T-cell abnormalities. We report the performance of the SCID screening program and the outcomes of infants who were identified.METHODS: Data that were reviewed and analyzed included demographics, nursery summaries, TREC and lymphocyte flow-cytometry values, and available follow-up, including clinical and genetic diagnoses, treatments, and outcomes.RESULTS: Infants with clinically significant T-cell lymphopenia (TCL) were successfully identified at a rate of 1 in 15 300 births. Of these, 50 cases of SCID, or 1 in 65 000 births (95% confidence interval 1 in 51 000-1 in 90 000) were found. Prompt treatment led to 94% survival. Infants with non-SCID TCL were also identified, diagnosed and managed, including 4 with complete DiGeorge syndrome who received thymus transplants. Although no cases of typical SCID are known to have been missed, 2 infants with delayed-onset leaky SCID had normal neonatal TREC screens but came to clinical attention at 7 and 23 months of age.CONCLUSIONS: Population-based TREC testing, although unable to detect immune defects in which T cells are present at birth, is effective for identifying SCID and clinically important TCL with high sensitivity and specificity. The experience in California supports the rapid, widespread adoption of SCID newborn screening.

The pulsed EPR methods of electron spin echo envelope modulation (ESEEM) and electron spin echoelectron nuclear double resonance (ESE-ENDOR) are used to investigate the proximity of exchangeable hydrogens around the paramagnetic S 2 -state Mn cluster of the photosystem II oxygen-evolving complex. Although ESEEM and ESE-ENDOR are both pulsed electron paramagnetic resonance techniques, the specific mechanisms by which nuclear spin transitions are observed are quite different. We are able to generate good simulations of both 1 H ESE-ENDOR and 2 H ESEEM signatures of exchangeable hydrogens at the S 2 -state cluster. The convergence of simulation parameters for both methods provides a high degree of confidence in the simulations. Several exchangeable protons-deuterons with strong dipolar couplings are observed. In the simulations, two of the close (Ϸ2.5 Å ) hydrogen nuclei exhibit strong isotropic couplings and are therefore most probably associated with direct substrate ligation to paramagnetic Mn. Another two of the close (Ϸ2.7 Å ) hydrogen nuclei show no isotropic couplings and are therefore most probably not contained in Mn ligands. We suggest that these proximal hydrogens may be associated with a Ca 2ϩ -bound substrate, as indicated in recent mechanistic proposals for O 2 formation.

The molybdenum nitrogenase, present in a diverse group of bacteria and archea, is the major contributor to biological nitrogen fixation. The nitrogenase active site contains an iron-molybdenum cofactor (FeMo-co) composed of 7Fe, 9S, 1Mo, one unidentified light atom, and homocitrate. The nifQ gene was known to be involved in the incorporation of molybdenum into nitrogenase. Here we show direct biochemical evidence for the role of NifQ in FeMo-co biosynthesis. As-isolated NifQ was found to carry a molybdenum-iron-sulfur cluster that serves as a specific molybdenum donor for FeMo-co biosynthesis. Purified NifQ supported in vitro FeMo-co synthesis in the absence of an additional molybdenum source. The mobilization of molybdenum from NifQ required the simultaneous participation of NifH and NifEN in the in vitro FeMo-co synthesis assay, suggesting that NifQ would be the physiological molybdenum donor to a hypothetical NifEN/NifH complex.nif ͉ iron-sulfur cluster ͉ Azotobacter vinelandii B iological nitrogen fixation performed by microorganisms that have nitrogenase(s) accounts for roughly two-thirds of the nitrogen fixed globally. Most nitrogen fixation is carried out by the activity of molybdenum nitrogenase (1), which is widely distributed in nature (2). The molybdenum-nitrogenase enzyme is composed of two proteins (3): a heterotetrameric NifDKprotein component (a 2 ␤ 2 dinitrogenase) and a homodimeric NifH-protein component (dinitrogenase reductase). NifDK contains one iron-molybdenum cofactor (FeMo-co) within the active site of each ␣-subunit (NifD), and one [8Fe-7S] P-cluster at the interface of the ␣-and ␤-subunits in each ␣␤ pair (4). NifH contains a [4Fe-4S] cluster bridging the two subunits and one site for Mg-ATP binding and hydrolysis in each subunit (5).FeMo-co is a unique cofactor ʈ composed of a [Mo-3Fe-3S] partial cubane bridged to a [4Fe-3S] partial cubane by three sulfur ligands and one unidentified light atom in the center of the cofactor (6). The molybdenum atom is also coordinated to the organic acid homocitrate. The biosynthesis of FeMo-co is a complex process that involves the activities of several nitrogen fixation (nif ) gene products that function as molecular scaffolds, enzymes, or escort proteins that carry FeMo-co precursors between assembly sites in the pathway (7,8). Following assembly, FeMo-co is inserted into apo-NifDK, a P-cluster-containing but FeMo-co-deficient form of NifDK that is matured into functional NifDK simply by FeMo-co insertion, to generate the mature dinitrogenase enzyme that is competent for nitrogen fixation.The NifEN scaffold protein is believed to function in the pathway as a central node to which an [Fe-S]-containing FeMo-co precursor, molybdenum, and homocitrate might converge to complete the assembly of FeMo-co (9-12). In this model, the [Fe-S]-containing FeMo-co precursor, NifB-co**, would be initially assembled by NifB and then transferred to NifEN for its conversion into the [Fe-S] VK-cluster (13,14). Homocitrate would be provided by the homocitrate-synthase ac...

Electron paramagnetic resonance studies at multiple frequencies (MF EPR) can provide detailed electronic structure descriptions of unpaired electrons in organic radicals, inorganic complexes, and metalloenzymes. Analysis of these properties aids in the assignment of the chemical environment surrounding the paramagnet and provides mechanistic insight into the chemical reactions in which these systems take part. Herein, we present results from pulsed EPR studies performed at three different frequencies (9, 31, and 130 GHz) on [Mn(II)(H 2 O) 6 ] 2+ , Mn(II) adducts with the nucleotides ATP and GMP, and the Mn(II)-bound form of the hammerhead ribozyme (MnHH). Through line shape analysis and interpretation of the zero-field splitting values derived from successful simulations of the corresponding continuous-wave and field-swept echodetected spectra, these data are used to exemplify the ability of the MF EPR approach in distinguishing the nature of the first ligand sphere. A survey of recent results from pulsed EPR, as well as pulsed electron-nuclear double resonance and electron spin echo envelope modulation spectroscopic studies applied to Mn(II)-dependent systems, is also presented. Mn-Containing Biological SystemsManganese operates as a cofactor in numerous proteins, serving both catalytic and structural roles [1][2][3][4]. Many Mn-dependent enzymes take advantage o f the rich redox chemistry available to the metal, accessing the +2, +3, +4, and perhaps even the +5 oxidation states during their turnover. For example, Mn-superoxide dismutase (MnSOD), which detoxifies the cell of the superoxide radical , cycles between the Mn(II) and Mn(III) oxidation states via the ping-pong type mechanism shown below [5][6][7][8][9].(1a) (1b) Other examples of such mononuclear redox-active enzymes include the manganese peroxidase responsible for lignin degradation by white-rot fungus [10][11][12]; a unique Mndependent form of lipoxygenase [13][14][15][16]; oxalate decarboxylase [17,18]; as well as an extradiol catechol dioxygenase [19][20][21]. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptIn order to help minimize kinetic and thermodynamic penalties associated with electron transfer events and the execution of chemical reactions, two or more metal centers can be coupled together to provide active sites capable of conducting multiple electrons while still operating within a physiologically accessible range of reduction potentials [22]. Only three such examples of redox-active polynuclear Mn enzymes are known: Mn-catalase that disproportionates hydrogen peroxide [23][24][25][26]; a Mn form of ribonucleotide reductase (Mn-RNR) [27,28]; and, arguably the most recognized Mn-dependent enzyme, the photosynthetic core of green plants, algae, and certain cyanobacteria termed photosystem II (PSII) [29,30]. Through a series of photoinitiated electron transfer events, oxidizing equivalents are stored on the tetranuclear Mn core of PSII -the oxygen-evolving complex (OEC) -which then extracts four electr...

We report CW-EPR, ESEEM, and structural NMR results, as well as DFT calculations, on model compounds relevant to the unusual cross-linked Tyr-His (YH) moiety at the active site of the heme-copper oxidases. CW-EPR spectra of an (15)N isotopically labeled 4-methyl-2-(4-methyl-imidazole-1-yl)-phenol radical are nearly identical to those of the natural abundance (14)N compound. We obtain good simulations of these EPR spectra without including hyperfine couplings to the nitrogen nuclei. This implies that the electron distribution of the radical is largely localized on the phenol ring with only a small amount of spin delocalized onto the nitrogens of the imidazole. Using three-pulse ESEEM spectroscopy, we have successfully detected the two imidazole ring nitrogens, one near the "exact cancellation" ESEEM condition and the other more weakly coupled. We assign these to the imino and amino nitrogens, respectively, based on DFT calculations performed on this radical species. The experimental results and the supporting density functional calculations clearly show that the imidazole substituent has only a minor effect on the electronic structure of the substituted phenol radical.