Catalytic conversion of nitrogen to ammonia by an iron model complex

Threefold symmetric Fe phosphine complexes have been used to model the structural and functional aspects of biological N₂ fixation by nitrogenases. Low-valent bridging Fe-S-Fe complexes in the formal oxidation states Fe(II)Fe(II), Fe(II)/Fe(I), and Fe(I)/Fe(I) have been synthesized which display rich spectroscopic and magnetic behavior. A series of cationic tris-phosphine borane (TPB) ligated Fe complexes have been synthesized and been shown to bind a variety of nitrogenous ligands including N₂H₄, NH₃, and NH₂-. These complexes are all high spin S = 3/2 and display EPR and magnetic characteristics typical of this spin state. Furthermore, a sequential protonation and reduction sequence of a terminal amide results in loss of NH₃ and uptake of N₂. These stoichiometric transformations represent the final steps in potential N₂ fixation schemes.

Treatment of an anionic FeN₂ complex with excess acid also results in the formation of some NH₃, suggesting the possibility of a catalytic cycle for the conversion of N₂ to NH₃ mediated by Fe. Indeed, use of excess acid and reductant results in the formation of seven equivalents of NH₃ per Fe center, demonstrating Fe mediated catalytic N₂ fixation with acids and protons for the first time. Numerous control experiments indicate that this catalysis is likely being mediated by a molecular species.

A number of other phosphine ligated Fe complexes have also been tested for catalysis and suggest that a hemi-labile Fe-B interaction may be critical for catalysis. Additionally, various conditions for the catalysis have been investigated. These studies further support the assignment of a molecular species and delineate some of the conditions required for catalysis.

Finally, combined spectroscopic studies have been performed on a putative intermediate for catalysis. These studies converge on an assignment of this new species as a hydrazido(2-) complex. Such species have been known on group 6 metals for some time, but this represents the first characterization of this ligand on Fe. Further spectroscopic studies suggest that this species is present in catalytic mixtures, which suggests that the first steps of a distal mechanism for N₂ fixation are feasible in this system.

A genetic and biochemical analysis of pre-mRNA splicing in Saccharomyces cerevisiae

Pre-mRNA splicing requires interaction of cis- acting intron sequences with trans -acting factors: proteins and small nuclear ribonucleoproteins (snRNPs). The assembly of these factors into a large complex, the spliceosome, is essential for the subsequent two step splicing reaction. First, the 5' splice site is cleaved and free exon 1 and a lariat intermediate (intron- exon2) form. In the second reaction the 3' splice site is cleaved the exons ligated and lariat intron released. A combination of genetic and biochemical techniques have been used here to study pre-mRNA splicing in yeast.

Yeast introns have three highly conserved elements. We made point mutations within these elements and found that most of them affect splicing efficiency in vivo and in vitro, usually by inhibiting spliceosome assembly.

To study trans -acting splicing factors we generated and screened a bank of temperature- sensitive (ts) mutants. Eleven new complementation groups (prp17 to prp27) were isolated. The four phenotypic classes obtained affect different steps in splicing and accumulate either: 1) pre-mRNA, 2) lariat intermediate, 3) excised intron or 4) both pre-mRNA and intron. The latter three classes represent novel phenotypes. The excised intron observed in one mutant: prp26 is stabilized due to protection in a snRNP containing particle. Extracts from another mutant: prpl8 are heat labile and accumulate lariat intermediate and exon 1. This is especially interesting as it allows analysis of the second splicing reaction. In vitro complementation of inactivated prp18 extracts does not require intact snRNPs. These studies have also shown the mutation to be in a previously unknown splicing protein. A specific requirement for A TP is also observed for the second step of splicing. The PRP 18 gene has been cloned and its polyadenylated transcript identified.

Electronic structure in five-coordinate complexes of nickel(II) with heavy-donor ligands

I.

Various studies designed to elucidate the electronic structure of the arsenic donor ligand, o-phenylenebisdimethylarsine (diarsine), have been carried out. The electronic spectrum of diarsine has been measured at 300 and 77˚K. Electronic spectra of the molecular complexes of various substituted organoarsines and phosphines with tetracyanoethylene have been measured and used to estimate the relative ionization potentials of these molecules.

Uv photolysis of arsines in frozen solution (96˚K) has yielded thermally labile, paramagnetic products. These include the molecular cations of the photolyzed compounds. The species (diars)⁺ exhibits hyper-fine splitting due to two equivalent ⁷⁵As(I=3/2) nuclei. Resonances due to secondary products are reported and assignments discussed.

Evidence is presented for the involvement of d-orbitals in the bonding of arsines. In (diars)⁺ there is mixing of arsenic “lone-pair” orbitals with benzene ring π-orbitals.

II.

Detailed electronic spectral measurements at 300 and 77˚K have been carried out on five-coordinate complexes of low-spin nickel(II), including complexes of both trigonal bipyramidal (TBP) and square pyramidal (SPY) geometry. TBP complexes are of the form NiLX⁺ (X=halide or cyanide,

L = Qƭ(CH₂)₃As(CH₃)₂]₃ or

P [hexagon - Q'CH₃] , Q = P, As,

Q’=S, Se).

The electronic spectra of these compounds exhibit a novel feature at low temperature. The first ligand field band, which is asymmetric in the room temperature solution spectrum, is considerably more symmetrical at 77˚K. This effect is interpreted in terms of changes in the structure of the complex.

The SPY complexes are of the form Ni(diars)₂X^z (X=CL, Br, CNS, CN, thiourea, NO₂, As). On the basis of the spectral results, the d-level ordering is concluded to be xy ˂ xz, yz ˂ z² ˂˂ x² - y². Central to this interpretation is identification of the symmetry-allowed ¹A₁ → ¹E (xz, yz → x² - y²) transition. This assignment was facilitated by the low temperature measurements.

An assignment of the charge-transfer spectra of the five-coordinate complexes is reported, and electronic spectral criteria for distinguishing the two limiting geometries are discussed.

Enzyme induction in Neurospora crassa

I. Studies on Nicotinamide Adenine Dinucleotide Glycohydrase (NADase)

NADase, like tyrosinase and L-amino acid oxidase, is not present in two day old cultures of wild type Neurospora, but it is coinduced with those two enzymes during starvation in phosphate buffer. The induction of NADase, like tyrosinase, is inhibited by puromycin. The induction of all three enzymes is inhibited by actinomycin D. These results suggest that NADase is synthesized de novo during induction as has been shown directly for tyrosinase. NADase induction differs in being inhibited by certain amino acids.

The tyrosinaseless mutant ty-1 contains a non-dialyzable, heat labile inhibitor of NADase. A new mutant, P110A, synthesizes NADase and L-amino acid oxidase while growing. A second strain, pe, fl;cot, makes NADase while growing. Both strains can be induced to make the other enzymes. These two strains prove that the control of these three enzymes is divisible. The strain P110A makes NADase even when grown in the presence of Tween 80. The synthesis of both NADase and L-amino acid oxidase by P110A is suppressed by complete medium. The theory of control of the synthesis of the enzymes is discussed.

II. Studies with EDTA

Neurospora tyrosinase contains copper but, unlike other phenol oxidases, this copper has never been removed reversibly. It was thought that the apo-enzyme might be made in vivo in the absence of copper. Therefore cultures were treated with EDTA to remove copper before the enzyme was induced. Although no apo-tyrosinase was detected, new information on the induction process was obtained.

A treatment of Neurospora with 0.5% EDTA pH 7, inhibits the subsequent induction during starvation in phosphate buffer of tyrosinase, L-amino acid oxidase and NADase. The inhibition of tyrosinase and L-amino acid oxidase induction is completely reversed by adding 5 x 10^-5M CaCl₂, 5 x 10^-4M CuSO₄, and a mixture of L-amino acids (2 x 10^-3M each) to the buffer. Tyrosinase induction is also fully restored by 5 x 10^-4M CaCl₂ and amino acids. As yet NADase has been only partially restored.

The copper probably acts by sequestering EDTA left in the mycelium and may be replaced by nickel. The EDTA apparently removes some calcium from the mycelium, which the added calcium replaces. Magnesium cannot replace calcium. The amino acids probably replace endogenous amino acids lost to the buffer after the EDTA treatment.

The EDTA treatment also increases permeability, thereby increasing the sensitivity of induction to inhibition by actinomycin D and allowing cell contents to be lost to the induction buffer. EDTA treatment also inhibits the uptake of exogenous amino acids and their incorporation into proteins.

The lag period that precedes the first appearance of tyrosinase is demonstrated to be a separate dynamic phase of induction. It requires oxygen. It is inhibited by EDTA, but can be completed after EDTA treatment in the presence of 5 x 10^-5M CaCl₂ alone, although no tyrosinase is synthesized under these conditions.

The time course of induction has an early exponential phase suggesting an autocatalytic mechanism of induction.

The mode of action of EDTA, the process of induction and the kinetics of induction are discussed.