The reduction by NADPH of the FAD and FMN redox centers in human cytochrome P450 reductase and its component domains has been studied by rapid-mixing, stopped-flow spectroscopy. Reduction of the isolated FAD-domain occurs in three kinetically resolvable steps. The first represents the rapid formation (>500 s(-)(1)) of a charge-transfer species between oxidized FAD and NADPH. This is followed by an isomerization ( approximately 200 s(-)(1)) to a second charge-transfer species, characterized by a more intense absorption in the long-wavelength region. The third step represents hydride transfer from NADPH to FAD and is accompanied by a change in the tryptophan fluorescence of the FAD-domain. Flavin reduction is reversible, and the observed rate of hydride transfer displays a complex dependence on NADPH concentration. Two-electron-reduced FAD-domain is active in electron transfer reactions with the isolated FMN domain through the formation of a weakly associating electron transfer complex. Reduction of the CPR by NADPH occurs without direct spectral evidence for the formation of charge-transfer species, although the presence of such species is inferred indirectly. Transfer of the first hydride ion leads to the accumulation of a blue di-semiquinoid species of the reductase, indicating rapid transfer of one electron to the FMN domain. The di-semiquinoid species decays on transfer of the second hydride ion. A third phase is seen following prolonged incubation with NADPH and is assigned to a series of equilibration reactions between different redox species of the enzyme as the system relaxes to its thermodynamically most stable state. As with the isolated FAD-domain, the first hydride transfer in the reductase shows a complex dependence on NADPH concentration. At high NADPH concentration, the observed rate of hydride transfer is slow (approximately 20 s(-1)), and this attenuated rate is attributed to the reversible formation of an less active complex resulting from the binding of a second molecule of NADPH. The kinetic data are discussed with reference to the potentiometric studies on the enzyme and its component domains presented in the preceding paper in this issue [Munro, A., Noble, M., Robledo, L., Daff, S., and Chapman, S. (2001) Biochemistry 40, 1956-1963].

The post-translationally modified peptide antibiotic nisin has been cleaved by a number of proteases and the fragments produced purified, characterised chemically, and assayed for activity in inhibiting the growth of Lactococcus lactis MG1614 and Micrococcus luteus NCDO8166. These results provide information on the importance of different parts of the nisin molecule for its growth-inhibition activity. Removal of the C-terminal five residues leads to approximately a 10-fold decrease in potency, while removal of a further nine residues, encompassing two of the lanthionine rings, leads to a 100-fold decrease. There are some differences between analogous fragments of nisin and subtilin, suggesting possible subtle differences in mode of action. Cleavage within, or removal of, lanthionine ring C essentially abolishes the activity of nisin. The fragment ulsin t-12 is inactive itself, and specifically antagonises the growth-inhibitory action of nisin. These results are discussed in lerms of current models for the mechanism of action of nisin.Key words: Nisin; Lantibiotic; Peptide antibiotic; I'roteolysis; Structure-activity relationships mation of voltage-dependent pores in biological membranes [8][9][10], although recent evidence indicates that the inhibition of the outgrowth of spores by nisin and subtilin takes place by a different mechanism [11,12]. We have been studying the structure-activity relationships in nisin and subtilin, by both genetic and chemical modification of the structure [12][13][14][15][16][17][18][19][20], with a view to understanding their mechanism of action and to developing new derivatives with desirable properties. We now report the preparation, characterisation and anti-bacterial activity of a number of proteolytic fragments of nisin and subtilin, which allow us to define the parts of the molecule most important for biological activity. Materials and methodsNisin and subtilin were prepared as previously described [14,15]; [Ser33]-nisin, in which the serine residue at position 33 has escaped processing to a dehydroalanine residue, was isolated as a minor component of commercial nisin. All proteases were obtained from Sigma Chemical Co., Poole, Dorset, UK. ~. IntroductionThe post-translationally modified peptide antibiotics known :ts 'lantibiotics' contain cyclic structures formed by lanthioaine or 3-methyl-lanthionine residues, and often also dehydroalanine and/or dehydrobutyrine residues [1]. The first lan-!ibiotic to be characterised was nisin, produced by strains of Lactococcus lactis carrying a transposon containing genes coding for the nisin precursor and for proteins involved in aisin biosynthesis and resistance [2][3][4][5]. Nisin has been quite videly used as a food preservative, notably in cheese and ,~ther dairy products and in canned vegetables, for some 30 .,ears [6,7]. It inhibits the growth of a wide range of Gram~ositive organisms, and also inhibits the germination and/or mtgrowth of spores of Bacillus and Clostridium species [6]. Fhe growth-inhibitory activity of nisi...

The binding of the substrates sodium laurate and sodium 12-bromolaurate to the heme-containing domain of Bacillus megaterium cytochrome P450 BM3 (CYP102) has been studied by measurement of the relaxation effects of the unpaired electrons of the heme iron on the protons of water and of the bound substrates. Substrate binding leads to a conversion of the heme iron from a low-spin to a high-spin state, as shown by changes in the optical spectrum. The relaxation measurements show that this is accompanied by expulsion of water from the sixth coordination position of the iron, the distance between the iron and the water protons increasing from 2.6 to 5.2 A. Corresponding relaxation measurements on the substrate protons lead to the determination of a number of distances between the iron and protons of the bound substrate and, hence, to information on the position and orientation of the substrate in the binding site. Laurate and 12-bromolaurate are found to bind in a very similar way, in an extended conformation with the carboxylate probably close to Arg47 and the other end of the chain 7.6-7.8 A from the heme iron. It is shown that laurate and pyridine can bind simultaneously to the P450 domain and that the iron-laurate distances in this ternary complex are not significantly different from those in the binary complex. These observations are compared with those on the substrate complex of cytochrome P450 cam, and their implications for structural changes involved in the catalytic cycle are discussed.

The serine/arginine-rich (SR) protein splicing factor 2/alternative splicing factor (SF2/ASF) has a role in splicing, stability, export and translation of messenger RNA. Here, we present the structure of the RNA recognition motif (RRM) 2 from SF2/ASF, which has an RRM fold with a considerably extended loop 5 region, containing a two-stranded b-sheet. The loop 5 extension places the previously identified SR protein kinase 1 docking sequence largely within the RRM fold. We show that RRM2 binds to RNA in a new way, by using a tryptophan within a conserved SWQLKD motif that resides on helix a1, together with amino acids from strand b2 and a histidine on loop 5. The linker connecting RRM1 and RRM2 contains arginine residues, which provide a binding site for the mRNA export factor TAP, and when TAP binds to this region it displaces RNA bound to RRM2.