We here show that the iron-molybdenum (FeMo)-cofactor of the nitrogenase alpha-70(Ile) molybdenum-iron (MoFe) protein variant accumulates a novel S = (1)/(2) state that can be trapped during the reduction of protons to H(2). (1,2)H-ENDOR measurements disclose the presence of two protons/hydrides (H(+/)(-)) whose hyperfine tensors have been determined from two-dimensional field-frequency (1)H ENDOR plots. The two H(+/)(-) have large isotropic hyperfine couplings, A(iso)( )() approximately 23 MHz, which shows they are bound to the cofactor. The favored analysis for these plots indicates that the two H(+/)(-) have the same principal values, which indicates that they are chemically equivalent. The tensors are further related to each other by a permutation of the tensor components, which indicates an underlying symmetry of binding relative to the cofactor. At present, no model for the structure of the iron-molybdenum (FeMo)-cofactor in the S = (1)/(2) state trapped during the reduction of H(+) can be shown unequivocally to satisfy all of the constraints generated by the ENDOR analysis. The data disfavors any model that involves protonation of sulfides, and thus suggests that the intermediate instead contains two chemically equivalent bound hydrides; it appears unlikely that these are terminal monohydrides.

This review focuses on recent developments elucidating the mechanism of the Mo-dependent nitrogenase. This enzyme, responsible for the majority of biological nitrogen fixation, is composed of two component proteins called the MoFe protein and the Fe protein. Recent progress in understanding the mechanism of this enzyme has focused on elucidating the structures of the active site metal clusters and of the proteins, understanding substrate interactions with the active site, defining the flow of electron transfer between the metal clusters, and defining the various roles of MgATP hydrolysis.

N2 binds to the active-site metal cluster in the nitrogenase MoFe protein, the FeMo-cofactor ([7Fe-9S-Mo-homocitrate-X]; FeMo-co) only after the MoFe protein has accumulated three or four electrons/protons (E3 or E4 states), with the E4 state being optimally activated. Here we study the FeMo-co 57Fe atoms of E4 trapped with the α-70Val→Ile MoFe protein variant through use of advanced ENDOR methods: ‘random-hop’ Davies pulsed 35 GHz ENDOR; difference Triple resonance; the recently developed Pulse-Endor-SaTuration and REcovery (PESTRE) protocol for determining hyperfine-coupling signs; and Raw-DATA (RD)-PESTRE, a PESTRE variant that gives a continuous sign readout over a selected radiofrequency range. These methods have allowed experimental determination of the signed isotropic 57Fe hyperfine couplings for five of the seven iron sites of the reductively activated E4 FeMo-co, and given the magnitude of the coupling for a sixth. When supplemented by the use of sum-rules developed to describe electron-spin coupling in FeS proteins, these 57Fe measurements yield both the magnitude and signs of the isotropic couplings for the complete set of seven Fe sites of FeMo-co in E4. In light of the previous findings that FeMo-co of E4 binds two hydrides in the form of (Fe-(μ-H−)-Fe) fragments, and that molybdenum has not become reduced, an ‘electron inventory’ analysis assigns the formal redox level of FeMo-co in E4 to that of the resting state (MN), with the four accumulated electrons residing on the two Fe-bound hydrides. Comparisons with earlier 57Fe ENDOR studies and electron inventory analyses of the bio-organometallic intermediate formed during the reduction of alkynes and the CO-inhibited forms of nitrogenase (hi-CO and lo-CO) inspire the conjecture that throughout the eight-electron reduction of N2 plus 2H+ to two NH3 plus H2, the inorganic core of FeMo-co cycles through only a single redox couple connecting the two formal redox levels: those associated with the resting state, MN, and the one-electron reduced state, MR. We further note that this conjecture might apply to other complex FeS enzymes.

Nitrogenase is the metalloenzyme that catalyzes the nucleotide-dependent reduction of N(2), as well as reduction of a variety of other triply bonded substrates, including the alkyne, acetylene. Substitution of the alpha-70(Val) residue in the nitrogenase MoFe protein by alanine expands the range of substrates to include short-chain alkynes not reduced by the unaltered protein. Rapid freezing of the alpha-70(Ala) nitrogenase MoFe protein during reduction of the alkyne propargyl alcohol (HC triple bond CH(2)OH; PA) traps an S = (1)/(2) intermediate state of the active-site metal cluster, the FeMo-cofactor. We have combined CW and pulsed (13)C ENDOR (electron-nuclear double resonance) with two quantitative 35 GHz (1,2)H ENDOR techniques, Mims pulsed ENDOR and the newly devised "stochastic field-modulated" ENDOR, to study this intermediate prepared with isotopically substituted ((13)C, (1,2)H) propargyl alcohol in H(2)O and D(2)O buffers. These measurements allow the first description of a trapped nitrogenase reduction intermediate. The S = (1)/(2) turnover intermediate generated during the reduction of PA contains the 3-carbon chain of PA and exhibits resolved (1,2)H ENDOR signals from three protons, two strongly coupled (H(a)) and one weakly coupled (H(b)); H(a)(c) originates as the C3 proton of PA, while H(a)(s) and H(b) are solvent-derived. The two H(a) protons have identical hyperfine tensors, despite having different origins. The equality of the (H(a)(s), H(a)(c)) hyperfine tensors strongly constrains proposals for the structure of the cluster-bound reduced PA. Through consideration of model structures found in the Cambridge Structural Database, we propose that the intermediate contains a novel bio-organometallic complex in which a reduction product of propargyl alcohol binds as a metalla-cyclopropane ring to a single Fe atom of the Fe-S face of the FeMo-cofactor that is composed of Fe atoms 2, 3, 6, and 7. Of the two most attractive structures, one singly reduced at C3 (4), the other being the doubly reduced allyl alcohol product (6), we tentatively favor 6 because of the "natural" assignment it affords for H(b).

The chemical mechanism for biological cleavage of the N(2) triple bond at ambient pressure and temperature has been the subject of intense study for many years. The site of substrate activation and reduction has been localized to a complex cofactor, called FeMo cofactor, yet until now the complexity of the system has denied information concerning exactly where and how substrates interact with the metal-sulfur framework of the active site. In this Account, we describe a combined genetic, biophysical, and biochemical approach that was used to provide direct and detailed information concerning where alternative alkyne substrates interact with FeMo cofactor during catalysis. The relevance and limitations of this work with respect to N(2) binding and reduction also are discussed.

Despite advances in treatments like chemotherapy and radiotherapy, metastatic cancer remains a leading cause of death for cancer patients. While many chemotherapeutic agents can efficiently eliminate cancer cells, long-term protection against cancer is not achieved and many patients experience cancer recurrence. Mobilizing and stimulating the immune system against tumor cells is one of the most effective ways to protect against cancers that recur and/or metastasize. Activated tumor specific cytotoxic T lymphocytes (CTLs) can seek out and destroy metastatic tumor cells and reduce tumor lesions. Natural Killer (NK) cells are a front-line defense against drug-resistant tumors and can provide tumoricidal activity to enhance tumor immune surveillance. Cytokines like IFN-γ or TNF play a crucial role in creating an immunogenic microenvironment and therefore are key players in the fight against metastatic cancer. To this end, a group of anthracyclines or treatments like photodynamic therapy (PDT) exert their effects on cancer cells in a manner that activates the immune system. This process, known as immunogenic cell death (ICD), is characterized by the release of membrane-bound and soluble factors that boost the function of immune cells. This review will explore different types of ICD inducers, some in clinical trials, to demonstrate that optimizing the cytokine response brought about by treatments with ICD-inducing agents is central to promoting anti-cancer immunity that provides long-lasting protection against disease recurrence and metastasis.

Nitrogenase catalyzes biological dinitrogen fixation, the reduction of N 2 to 2NH 3 . Recently, the binding site for a non-physiological alkyne substrate (propargyl alcohol, HC'C-CH 2 OH) was localized to a specific Fe-S face of the FeMo-cofactor approached by the MoFe protein amino acid ␣-70Val . Here we provide evidence to indicate that the smaller alkyne substrate acetylene (HC'CH), the physiological substrate dinitrogen, and its semi-reduced form hydrazine (H 2 N-NH 2 ) interact with the same Fe-S face of the FeMo-cofactor. Hydrazine is a relatively poor substrate for the wild-type (␣-70 Val ) MoFe protein. Substitution of the ␣-70Val residue by an amino acid having a smaller side chain (alanine) dramatically enhanced hydrazine reduction activity. Conversely, substitution of ␣-70Val by an amino acid having a larger side chain (isoleucine) significantly lowered the capacity of the MoFe protein to reduce dinitrogen, hydrazine, or acetylene.

Biological nitrogen fixation, the conversion of atmospheric N2 to NH3, is an essential process in the global biogeochemical cycle of nitrogen that supports life on Earth. Most of the biological nitrogen fixation is catalyzed by the molybdenum nitrogenase, which contains at its active site one of the most complex metal cofactors known to date, the iron-molybdenum cofactor (FeMo-co). FeMo-co is composed of 7Fe, 9S, Mo, R-homocitrate, and one unidentified light atom. Here we demonstrate the complete in vitro synthesis of FeMo-co from Fe 2؉ , S 2؊ , MoO4 2؊ , and R-homocitrate using only purified Nif proteins. This synthesis provides direct biochemical support to the current model of FeMo-co biosynthesis. A minimal in vitro system, containing NifB, NifEN, and NifH proteins, together with Fe 2؉ , S 2؊ , MoO4 2؊ , R-homocitrate, S-adenosyl methionine, and Mg-ATP, is sufficient for the synthesis of FeMo-co and the activation of apo-dinitrogenase under anaerobic-reducing conditions. This in vitro system also provides a biochemical approach to further study the function of accessory proteins involved in nitrogenase maturation (as shown here for NifX and NafY). The significance of these findings in the understanding of the complete FeMo-co biosynthetic pathway and in the study of other complex Fe-S cluster biosyntheses is discussed.FeMo-co ͉ nitrogen fixation ͉ nif ͉ Fe-S proteins B iological nitrogen fixation, the conversion of atmospheric N 2 to NH 3 , is an essential process of the global biogeochemical cycle of nitrogen that supports life on Earth (1). Biological nitrogen fixation provides an alternative solution to the heavy use of chemically synthesized nitrogen fertilizers and, thus, is important to achieve sustainable agricultural practices (2). The major part of biological nitrogen fixation is catalyzed by the molybdenum nitrogenase enzyme according to the following reaction: N 2 ϩ 8e Ϫ ϩ 16 MgATP ϩ 8 H ϩ 3 2 NH 3 ϩ H 2 ϩ 16 MgADP ϩ 16 Pi (3). Remarkably, this reaction produces H 2 as a by-product and has regained attention over the last few years as a promising source of clean energy (4). The molybdenum nitrogenase has two component proteins: dinitrogenase (NifDK) and dinitrogenase reductase (NifH). Dinitrogenase carries at its active site one of the most complex biological metalloclusters known to date, the iron-molybdenum cofactor (FeMo-co), composed of seven Fe, nine S, one Mo, one R-homocitrate, and one light unidentified atom (5, 6).The systematic phenotypic analyses of Azotobacter vinelandii and Klebsiella pneumoniae strains with mutations in nitrogen fixation (nif ) genes and the subsequent biochemical analyses have led to the identification of at least 11 genes (nifUS-BQ-ENX-V-H-Y and nafY) encoding proteins involved in FeMo-co biosynthesis and its insertion into a FeMo-co-deficient apoNifDK protein (7-9). Fig. 1 depicts an integrative model of the current understanding of the pathway for FeMo-co biosynthesis. It is important to emphasize that this model is mostly based on the ability to isolate nif muta...