Flavins are very versatile coenzymes, functioning with considerable efficiency in a wide variety of enzymic reactions involving either two-electron or one-electron transfers, with both functions often catalysed by the same enzyme. They are responsible for catalysing the dehydrogenation of many different types of compounds, including dithiols, reduced nicotinamide nuclwtides, alcohols and a-hydroxy acids, amines and a-amino acids, and even saturated CC bonds, provided that a suitable activating group such as a carbonyl residue is situated a/% to the bond to be oxidized. In the process of catalysing these dehydrogenation reactions, the flavin is itself reduced, and, in order to function catalytically, the oxidized form must be regenerated at the expense of reduction of some acceptor. The acceptor may be in some cases the oxidized form of the same type of compound that serves as reducing substrate, e.g. it might be a disulphide, an oxidized nicotinamide nucleotide or an unsaturated compound such as fumarate or crotonyl-CoA. In such a case the enzyme might conveniently be classified as a transhydrogenase and subclassified as a CC , C-S, C-N or N-N transhydrogenase, depending on the nature of the atoms acting as hydrogen donor and hydrogen acceptor (Hemmerich & Massey, 1979). In most cases, however, the acceptor molecule will be molecular 0, or another redox protein, such as an iron-sulphur protein or a cytochrome. In the latter cases the flavoprotein necessarily acts as a mediator between two-electron and one-electron transfers. Flavoproteins fill a unique spot in biochemistry with this capacity. An equal richness of possibilities exists in the reactions of different flavoproteins with molecular 0,. In some cases one-electron transfer is carried out, with superoxide (02-) and flavin semiquinone as the immediate products of the reaction. In other cases a direct two-electron reduction of 0, to H,O, appears to occur, and in another class of flavoproteins, the mono-oxygenases, one atom of the 0, molecule is incorporated into H 2 0 and the other is incorporated into another substrate of the enzyme, to form an oxygenated product. Functional classification offlavoproteins The types of reactivity described above have led us (Hemmerich & Massey, 1979) to a functional classification of simple flavoproteins, in which five major divisions may be recognized. (The complex metal-and haem-containing flavoproteins also fit into this classification, but are excluded from the present discussion.) C1a.w 1 (a): Carbon-carbon transhydrogenases. Enzymes this class catalyse two-electron transfer between carbon centres, such as between nicotinamide nuclwtides acting both as redox donors and acceptors or between nicotinamide nucleotide and acyl/enoyl-CoA. Class I (b): Carbon-sulphur transhydrogenases. This class of enzymes catalyses two-electron transfer between nicotinamide nuclwtide and disulphiddithiol pairs. Typical examples are lipoyl dehydrogenase and gluthathione reductase. Class I (c): Carbon-nitrogen transhydrogenases. Enzymes in thi...

Deazaflavins have been found to act as potent catalysts in the photoreduction of flavoproteins in the presence of EDTA and other "photosubstrates". In distinction to the catalysis brought about by normal flavins which involves dark reaction of the photoreduced flavin catalyst, the mechanism of the catalysis by deazaflavins has been shown to involve unstable, strongly reducing radicals which are generated by photolysis of a preformed covalent dimer. By this new method it is possible to reduce not only flavoproteins but a variety of other redox proteins, including heme proteins and iron-sulfur proteins. By virtue of its great catalytic efficiency, it is possible to employ concentrations of deazaflavin sufficiently low as not to interfere with the spectral evaluation of the reduced proteins obtained.

Abbreviations used in this paper: CENP, centromere protein; FCS, fl uorescence correlation spectroscopy; mRFP, monomeric red fl uorescent protein; PCNA, proliferating cell nuclear antigen.The online version of this paper contains supplemental material.

PML nuclear bodies (NBs) are involved in the regulation of key nuclear pathways but their biochemical function in nuclear metabolism is unknown. In this study PML NB assembly dynamics were assessed by live cell imaging and mathematic modeling of its major component parts. We show that all six nuclear PML isoforms exhibit individual exchange rates at NBs and identify PML V as a scaffold subunit. SP100 exchanges at least five times faster at NBs than PML proteins. Turnover dynamics of PML and SP100 at NBs is modulated by SUMOylation. Exchange is not temperature-dependent but depletion of cellular ATP levels induces protein immobilization at NBs. The PML-RARα oncogene exhibits a strong NB retention effect on wild-type PML proteins. HIPK2 requires an active kinase for PML NB targeting and elevated levels of PML IV increase its residence time. DAXX and BLM turn over rapidly and completely at PML NBs within seconds. These findings provide a kinetics model for factor exchange at PML NBs and highlight potential mechanisms to regulate intranuclear trafficking of specific factors at these domains.

Heterochromatin protein 1 (HP1) is a conserved nonhistone chromosomal protein with functions in euchromatin and heterochromatin. Here we investigated the diffusional behaviors of HP1 isoforms in mammalian cells. Using fluorescence correlation spectroscopy (FCS) and fluorescence recovery after photobleaching (FRAP) we found that in interphase cells most HP1 molecules (50 -80%) are highly mobile (recovery halftime: t 1/2 Ϸ 0.9 s; diffusion coefficient: D Ϸ 0.6 -0.7 m 2 s ؊1 ). Twenty to 40% of HP1 molecules appear to be incorporated into stable, slow-moving oligomeric complexes (t 1/2 Ϸ 10 s), and constitutive heterochromatin of all mammalian cell types analyzed contain 5-7% of very slow HP1 molecules. The amount of very slow HP1 molecules correlated with the chromatin condensation state, mounting to more than 44% in condensed chromatin of transcriptionally silent cells. During mitosis 8 -14% of GFP-HP1␣, but not the other isoforms, are very slow within pericentromeric heterochromatin, indicating an isoform-specific function of HP1␣ in heterochromatin of mitotic chromosomes. These data suggest that mobile as well as very slow populations of HP1 may function in concert to maintain a stable conformation of constitutive heterochromatin throughout the cell cycle. INTRODUCTIONThe genomic DNA within the eukaryotic nucleus is organized into structurally distinct domains that regulate gene expression and chromosome behavior (Lamond and Earnshaw, 1998). Chromosomes are composed of two types of domains: heterochromatin and euchromatin (Cohen and Lee, 2002;Grewal and Moazed, 2003). Constitutive heterochromatic domains at centromeres and telomeres consist of repetitive DNA and are largely transcriptionally silent. Euchromatin defines the gene-rich and transcriptionally active region of the cell nucleus (Grewal and Elgin, 2002). Heterochromatin mediates many diverse functions in the cell nucleus, including centromere function, gene silencing, regulation of gene expression, and nuclear organization. At centromeres, heterochromatin is required for proper sister chromatid cohesion and mitotic segregation (Bernard et al., 2001;Peters et al., 2001; Nonaka et al., 2002;Hall et al., 2003). Smaller heterochromatin domains are involved in epigenetic regulation of gene expression during development and cellular differentiation (Cavalli, 2002;Grewal and Moazed, 2003). Heterochromatic inactivation of one of the two X chromosomes, giving rise to the Barr body, is essential in dosage compensation in somatic cells of female mammals (Avner and Heard, 2001). The link between heterochromatin and transcriptional silencing has been firmly established by detailed analysis of the phenomenon PEV (position effect variegation), in which a gene is silenced by positioning it abnormally close to heterochromatin (Wallrath and Elgin, 1995).The establishment of heterochromatin requires the physical coupling of histone-modifying activities and structural proteins at specific genomic sites (Richards and Elgin, 2002). The "histone code" hypothesis predicts tha...