P450cam from Pseudomonas putida is the best-characterized member of the vast family of cytochrome P450s, and it has long been believed to have a more rigid and closed active site relative to other P450s. Here we report X-ray structures of P450cam crystallized in the absence of substrate and at high and low [K + ]. The camphor-free structures are observed in a distinct open conformation characterized by a water-filled channel created by the retraction of the F and G helices, disorder of the B′ helix and loss of the K + binding site. Crystallization in the presence of K + alone does not alter the open conformation, while crystallization with camphor alone is sufficient for closure of the channel. Soaking crystals of the open conformation in excess camphor does not promote camphor binding or closure, suggesting resistance to conformational change by the crystal lattice. This open conformation is remarkably similar to that seen upon binding large tethered substrates, showing that it is not the result of a perturbation by the ligand. Re-dissolved crystals of the open conformation are observed as a mixture of P420 and P450 forms, which is converted to the P450 form upon addition of camphor and K + . These data reveal that P450cam can dynamically visit an open conformation that allows access to the deeply buried active site without being induced by substrate or ligand.Cytochrome P450s are ubiquitous heme monooxygenases that activate O 2 for oxygen atom insertion into a wide variety of substrates (1). Over 10000 forms of P450s have been identified in bacteria, archaea, plants, fungi, and all higher eukaryotes (2). Examples include major drug metabolizing enzymes of the liver, and biosynthetic enzymes involved in steroid and prostaglandin pathways. These enzymes all share a common protein fold first observed for the camphor metabolizing P450cam from Pseudomonas putida (3,4).The large diversity in the specificity or promiscuity of P450s is believed to be due largely to variations in the structure of the substrate binding channel, which is defined by elements including the F and G helices, FG loop and regions near the B′ helix, which fold over and around the heme and the I helix to enclose the substrate and position it for attack by the ferryl heme center. Sequence variation of these elements result in P450s with structurally distinct substrate binding channels, and large differences in these regions have been observed in the structures of bacterial (5), microsomal (6), and mitochondrial P450s (7), supporting the view that the different conformations of the F, G and B′ helices are responsible for the large diversity in size, shape and specificity of the substrate binding cavity. † This work was supported by grant GM41049 from the NIH. ‡ The atomic coordinate and structures factors were deposited in the Protein Data Bank under the codes 3L61 (substrate-free, 200 mM P450cam is the most well-characterized of all P450s, and in many ways it has served as the archetypal model for all P450s, particularly those with high s...

A central aspect of aging research concerns the question as to when individuality in lifespan arises 1. We have now discovered that a transient increase in reactive oxygen species (ROS), which occurs naturally during early development in a subpopulation of synchronized Caenorhabditis elegans, sets processes into motion that increase stress resistance, improve redox homeostasis and ultimately prolong lifespan in those animals. We find that these effects are linked to the global ROS-mediated decrease in developmental histone H3K4me3 levels. Studies in HeLa cells confirmed that global H3K4me3 levels are ROS-sensitive, and that depletion of H3K4me3 levels increases stress resistance in mammalian cell cultures. In vitro studies identified the Set1/MLL histone methyltransferase as the redox sensitive unit of the H3K4-trimethylating COMPASS complex. Our findings imply a novel link between early-life events, ROS-sensitive epigenetic marks, stress resistance and lifespan.

Sgt1 has been identified as a subunit of both core kinetochore and SCF (Skp1-Cul1-F-box) ubiquitin ligase complexes and is also implicated in plant disease resistance. Sgt1 has two putative HSP90 binding domains, a tetratricopeptide repeat and a p23-like CHORD and Sgt1 (CS) domain. Using NMR spectroscopy, we show that only the CS domain of human Sgt1 physically interacts with HSP90. The tetratricopeptide repeat domain does not bind to either HSP90 or HSP70. Determination of the three-dimensional structure showed that the Sgt1-CS domain shares the same ␤-sandwich fold as p23 but lacks the last highly conserved ␤-strand in p23. Analysis of the structures of Sgt1-CS and p23 revealed a similar charge distribution on one of two opposing surfaces that suggests that it is the binding region for HSP90 in Sgt1. Although ATP is absolutely required for p23 binding to HSP90, Sgt1 binds to HSP90 also in the absence of the non-hydrolyzable analog ATP␥S. Our findings suggest the CS domain is a binding module for HSP90 distinct from p23-like domains, which implies that Sgt1 and related proteins function in recruiting heat shock protein activities to multiprotein assemblies. Heat shock protein 90 (HSP90)1 is a molecular chaperone important for protein folding. HSP90 is different from other chaperones because most of its substrates are related to signal transduction (1). Recent studies also suggest that HSP90 plays a role in protein quality control where it facilitates the polyubiquitination and degradation of substrates through interaction with the co-chaperone C terminus of HSC70-interacting protein (CHIP) (2). Thus, HSP90 can be involved in protein regulation in quite different ways depending on the cellular context.Very recently, it has been reported that Sgt1 interacts with HSP90 (3-5). Sgt1 was originally identified as a suppressor of the G 2 allele of Skp1 and was found to be important for both the G 1 /S and G 2 /M transitions in the cell cycle (6). The G 2 /M transition involves activation of the kinetochore. Sgt1 was shown to be required for the activation of the kinetochore core complex CBF3 and to physically interact with Skp1, one component of CBF3 (6). Sgt1 also physically interacts with Skp1-Cul1-F-box (SCF) E3 ubiquitin ligase complexes through interaction with Skp1. Moreover, a yeast Sgt1 mutant was defective in Sic1 degradation through ubiquitination (6).Sequence analysis of Sgt1 proteins from yeast, human, barley, rice, and Arabidopsis shows three conserved domains (tetratricopeptide repeat (TPR), CHORD-containing proteins and Sgt1 (CS), and Sgt1-specific (SGS)) and two variable regions (VR1 and VR2) (7). TPR domains are known as heat shock protein binding domains. The SGS domain was shown to interact with S100 calcium-binding proteins (8). The CS domain has high sequence homology with p23 and is also known as a p23-like domain (9). p23 has been shown to physically and functionally interact with HSP90, apparently serving in a role as co-chaperone. Recently, HSP90 was shown to be an essential factor required for...

High-valent iron-oxo species are thought to be intermediates in the catalytic cycles of oxygenases and peroxidases. An attractive route to these iron-oxo intermediates involves laser flash-quench oxidation of ferric hemes, as demonstrated by our work on the ferryl (compound II) and ferryl porphyrin radical cation (compound I) intermediates of horseradish peroxidase. Extension of this work to include cytochrome P450-BM3 (CYP102A1) has required covalent attachment of a Ru II photosensitizer to a nonnative cysteine near the heme (Ru II K97C -Fe III P450 ), in order to promote electron transfer from the Fe III porphyrin to photogenerated Ru III . The Ru II K97C -Fe III P450 conjugate was structurally characterized by X-ray crystallography (2.4 Å resolution; Ru-Fe distance, 24 Å). Flash-quench oxidation of the ferric-aquo heme produces an Fe IV -hydroxide species (compound II) within 2 ms. Difference spectra for three singly oxidized P450-BM3 intermediates were obtained from kinetics modeling of the transient absorption data in combination with generalized singular value decomposition analysis and multiexponential fitting.ruthenium bipyridine | enzyme catalysis T he cytochromes P450 constitute a superfamily of thiolateligated heme enzymes so named because the Soret absorption band in their CO-bound derivatives peaks near 450 nm. These monooxygenases catalyze a dazzling array of regio-and stereospecific oxidation reactions, including the hydroxylation of aliphatic and aromatic hydrocarbons and the epoxidation of alkenes (1, 2). P450s take two reducing equivalents from NAD(P)H and deliver one atom from dioxygen to the organic substrate; the other oxygen atom is released as water. The consensus mechanism for P450 catalysis (Fig. 1) implicates a ferryl porphyrin radical cation [compound I (CI): Fig. 1, intermediate 6] as the active oxygenating agent (3), but this elusive species has not been observed in P450 under single-turnover or steady-state catalytic conditions. In the postulated mechanism, CI is proposed to abstract a hydrogen atom from the substrate to form transient Fe IV -hydroxide complex (compound II, CII), followed by radical recombination to produce oxygenated product (4, 5). Mechanistic studies of P450 catalysis in cryogenic matrices have suggested that the barrier to formation of CI (5 → 6) is higher than that for its reaction with substrate (6 → 7 → 1) (6). Consequently, recent efforts have focused on alternate routes to P450 CI that bypass the hydroperoxide intermediate (5). One approach employs generation of CII using peroxynitrite, followed by laser flash photolysis to yield CI (7). This technique has been used in studies of the spectra and reactivity of the putative CI species, but the interpretation of the results remains open to question.In earlier work, we employed ½RuðbpyÞ 3 2þ (bpy ¼ 2;2 0 -bipyridine) in a bimolecular flash-quench photochemical oxidation protocol to generate CII and CI in horseradish peroxidase (HRP) and the heme octapeptide from cyctochrome c (MP8) (8, 9). This approach was uns...

Mixed lineage leukemia (MLL) family histone methyltransferases are enzymes that deposit histone H3 Lys4 (K4) mono-/di-/tri-methylation and regulate gene expression in mammals. Despite extensive structural and biochemical studies, the molecular mechanisms whereby the MLL complexes recognize histone H3K4 within nucleosome core particles (NCPs) remain unclear. Here we report the single-particle cryo-electron microscopy (cryo-EM) structure of the NCP-bound human MLL1 core complex. We show that the MLL1 core complex anchors to the NCP via the conserved RbBP5 and ASH2L, which interact extensively with nucleosomal DNA and the surface close to the N-terminal tail of histone H4. Concurrent interactions of RbBP5 and ASH2L with the NCP uniquely align the catalytic MLL1SET domain at the nucleosome dyad, thereby facilitating symmetrical access to both H3K4 substrates within the NCP. Our study sheds light on how the MLL1 complex engages chromatin and how chromatin binding promotes MLL1 tri-methylation activity.