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Nitrogenase is a globally important enzyme that catalyses the reduction of atmospheric dinitrogen into ammonia and is thus an important part of the nitrogen cycle. The nitrogenase enzyme is composed of a catalytic molybdenum–iron protein (MoFe protein) and a protein containing an [Fe4–S4] cluster (Fe protein) that functions as a dedicated ATP-dependent reductase. The current understanding of electron transfer between these two proteins is based on stopped-flow spectrophotometry, which has allowed the rates of complex formation and electron transfer to be accurately determined. Surprisingly, a total of four Fe protein molecules are required to saturate one MoFe protein molecule, despite there being only two well-characterized Fe-protein-binding sites. This has led to the conclusion that the purified Fe protein is only half-active with respect to electron transfer to the MoFe protein. Studies on the electron transfer between both proteins using rapid-quench EPR confirmed that, during pre-steady-state electron transfer, the Fe protein only becomes half-oxidized. However, stopped-flow spectrophotometry on MoFe protein that had only one active site occupied was saturated by approximately three Fe protein equivalents. These results imply that the Fe protein has a second interaction during the initial stages of mixing that is not involved in electron transfer.

Introduction It is now appropriate to discuss fine details of the mechanism of action of xanthine oxidase because of the extensive spectroscopic and kinetic evidence available; this is especially so since this has recently been supplemented by X-raycrystallographic data. Much of the information has been reviewed comprehensively [1,2] and is also discussed in this volume [3,4]. In this article we intend to concentrate on the role of electron nuclear double resonance (ENDOR) spectroscopy in determining aspects of the structure of intermediates in the mechanism, with particular reference to details of the evidence for Mo-C bonds, since there may be misunderstandings in the literature concerning how we interpreted our results [S]. We go on to consider some of the various hypotheses concerning the mechanisms that are currently being debated.

Engagement of the B-cell antigen receptor (BCR) induces the activation of various transcription factors, including NFAT (nuclear factor of activated T-cells) and NF-kappaB (nuclear factor kappaB), which participate in long-term biological responses such as proliferation, survival and differentiation of B-lymphocytes. We addressed the biochemical basis of this process using the DT40 chicken B-cell lymphoma. We discovered that Bruton's tyrosine kinase (BTK) and phospholipase C-gamma2 (PLC-gamma2) are required to activate NFAT and NF-kappaB, and to produce the lipid second messenger diacylglycerol in response to BCR cross-linking. Therefore the functional integrity of the BTK/PLC-gamma2/diacylglycerol signalling axis is crucial for BCR-directed activation of both transcription factors NFAT and NF-kappaB.

Nitrogenase comprises two proteins, each of which has very similar properties when isolated from any of the diazotrophs studied so far. Both exhibit e.p.r. signals in the resting state and during enzyme turnover. The larger one is generally called the MoFe protein, although other names are sometimes used (e.g. dinitrogenase; Hageman & Burris, 1978), and the most active preparations contain 2 Mo, 32 f 3 Fe and 30 S2atoms in an a2P2 molecule of 220 000 daltons. The smaller protein, the Fe protein (or dinitrogenase reductase; Hageman & Burris, 1978), is generally believed t o contain 4 Fe and 4 S2atoms in a y2 molecule of 66 000 daltons, although Haaker et al. (1984) have recently suggested that these metal and sulphide analyses may be in error. In this paper the nomenclature of Eady et al. (1972) will be used for the two proteins as isolated from individual diazotrophs, so that they are referred to by using a capital letter for the genus and a lower case letter for the species, followed by the number 1 for the MoFe protein and 2 for the Fe protein; thus, for example, MoFe protein isolated from Klebsiella pneumoniae is Kpl and the Fe protein isolated from Azotobacter vinelandii is Av2. The enzyme catalyses the reduction of N, t o 2 N H 3 (eqn. 1) with concomitant reduction of H+ t o H2 and hydrolysis of MgATP t o MgADP plus inorganic phosphate.