Electron-Induced Enzyme Activation
2006; Elsevier BV; Volume: 14; Issue: 1 Linguagem: Inglês
10.1016/j.str.2005.12.002
ISSN1878-4186
Autores Tópico(s)Electrochemical sensors and biosensors
ResumoIn this issue of Structure, Echalier et al., 2006Echalier A. Goodhew C.F. Pettigrew G.W. Fulop V. Structure. 2006; 14 (this issue): 107-117Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar report structures of a bacterial diheme cyctochrome c peroxidase in an oxidized inactive and an activated reduced state. The two structures give insight into the activation at one heme through reduction of the other. In this issue of Structure, Echalier et al., 2006Echalier A. Goodhew C.F. Pettigrew G.W. Fulop V. Structure. 2006; 14 (this issue): 107-117Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar report structures of a bacterial diheme cyctochrome c peroxidase in an oxidized inactive and an activated reduced state. The two structures give insight into the activation at one heme through reduction of the other. Members of the ubiquitous family of peroxidases play an important role in protecting organisms from damage by reactive oxygen species such as hydrogen peroxide both in standard conditions and under oxidative stress. The family can be separated into three classes based on sequence similarity. Class I enzymes are the intracellular peroxidases of prokaryotic origin exemplified by yeast cytochrome c peroxidase that have a single covalently attached heme group. Class II enzymes are secreted fungal peroxidases, e.g., lignin peroxidase, and class III peroxidases are found in higher plants and include horseradish peroxidase. Classes II and III use small organic molecules as a source of electrons for the reaction, whereas the cytochrome c peroxidases (enzyme classification 1.11.1.5) in class I stand apart because they are very specific for their target, cytochrome c (Yonetani, 1976Yonetani T. Academic Press. 1976; 13: 345-361Google Scholar). Yeast CCP, the first cytochrome c peroxidase identified (Altshul et al., 1940Altshul A.M. Abrams R. Hogness T.R. J. Biol. Chem. 1940; 136: 777-794Google Scholar), has become the paradigm of heme peroxidases. The enzyme reacts via a semistable enzyme intermediate, compound I, where the iron atom is in the Fe(IV) oxidation state and a semistable radical is formed on an amino acid side chain. In contrast, the bacterial Paracoccus pantotrophus (pap) (previously P. denitrificans [pad CCP], reclassified in Rainey et al., 1999Rainey F.A. Kelly D.P. Stackebrandt E. Burghardt J. Hiraishi A. Katayama Y. Wood A.P. Int. J. Syst. Bacteriol. 1999; 49: 645-651Crossref PubMed Scopus (142) Google Scholar) cytochrome c peroxidase is representative of a class of peroxidases that work without the need to form a semistable free radical for catalysis. They are found in the periplasmic space where they catalyze the reaction 2H+ + H2O2 + 2 cyt-c(Fe2+) → 2 cyt-c(Fe3+) + 2H2O. The bacterial CCP from Pseudomonas aeruginosa (Psa) was the first to be biochemically characterized (Ellfolk and Soininen, 1970Ellfolk N. Soininen R. Acta Chem. Scand. A. 1970; 24: 2126-2136Crossref Google Scholar) and to have its structure solved (Fulop et al., 1995Fulop V. Ridout C.J. Greenwood C. Hajdu J. Structure. 1995; 3: 1225-1233Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). Bacterial CCPs use two domains, each reminiscent of a class I c-type cytochrome with a covalently linked heme, to store the two oxidizing equivalents in the high-energy high-oxidation (compound I) form of the enzyme. Though chemically identical, the two hemes display very different spectral, biochemical, electrochemical, and physiological properties. One is in a low-spin state and, by analogy to the other peroxidases, is the peroxidatic site (P site) where H2O2 binds. The second heme is in equilibrium between high- and low-spin states and is the electron receiving site (E site), accepting the electrons from the physiological donor, e.g., cytochrome c550. The enzyme reaction thus requires intramolecular electron transfer between the two hemes and is potentially a tractable system for studying the mechanism of electron transfer. Bacterial CCPs are commonly isolated in an inactive oxidized state and are not able to bind hydrogen peroxide until activated in the presence of Ca2+. In this state, the P site heme is 6-coordinated with 2 histidine ligands, and the E site heme is also 6-coordinated. The exception to this is the Nitrosomonas europaea (Nep) CCP, which is active as isolated. The structure of this enzyme (Shimizu et al., 2001Shimizu H. Schuller D.J. Lanzilotta W.N. Sundaramoorthy M. Arciero D.M. Hooper A.B. Poulos T.L. Biochemistry. 2001; 40: 13483-13490Crossref PubMed Scopus (73) Google Scholar) therefore gave a first insight into the ligand binding P site in an active state with only one His ligand, but not into the process by which the other bacterial CCPs are activated. In 2004, Romao and colleagues published structures of CCP from Pseudomonas Nautica achieved by crystallization of oxidized enzyme at pH 4 (Ca2+ absent) and pH 5.3 (Ca2+ present) (Dias et al., 2004Dias J.M. Alves T. Bonifacio C. Pereira A.S. Trincao J. Bourgeois D. Moura I. Romao M.J. Structure. 2004; 12: 961-973Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). The principle differences between the two structures were in the region of the calcium binding site, where minor rearrangements occur, and in the ligation of the P site heme, which, like the nep structure, has five ligands at pH 5.3 and is therefore active-like. Neither form could support reduction except by X-rays at 100 K where proteins are immobilized (Weik et al., 2001Weik M. Ravelli R.B.G. Silman I. Sussman J.L. Gros P. Kroon J. Protein Sci. 2001; 10: 1953-1961Crossref PubMed Scopus (59) Google Scholar), implying that the conformational changes required on activation could not be supported in the crystal lattice. The current paper (Echalier et al., 2006Echalier A. Goodhew C.F. Pettigrew G.W. Fulop V. Structure. 2006; 14 (this issue): 107-117Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar) describes an alternative approach, obtaining both active and inactive states at a physiologically relevant pH, which show more substantial differences between oxidized and reduced states. The inactive form was crystallized as isolated. To achieve an unambiguous structure of the active state, the enzyme was crystallized in an anaerobic environment, in the presence of ascorbate. A chain of events is proposed to explain activation (Figure 1), triggered when an incoming electron reduces the E site heme, which is transmitted by conformational change(s) to the P site heme, leading to changes in the environment at the low potential heme to allow substrate access. A change in conformation of the E heme propionate following reduction of the heme is proposed to release a flexible loop (residues 225–257) (1) that takes up a new position at the interface between the two domains. This in turn leads to the movement of a second loop (residues 105–132) (2) away from this interface and into the vicinity of the P site. The final stage is release of the histidine sixth ligand (His85) at the P site and relocation of the his-ligand loop (80–92) (3) away from the P site, where it blocks access to the dimer interface. This “out” conformation is similar to that previously observed (Shimizu et al., 2001Shimizu H. Schuller D.J. Lanzilotta W.N. Sundaramoorthy M. Arciero D.M. Hooper A.B. Poulos T.L. Biochemistry. 2001; 40: 13483-13490Crossref PubMed Scopus (73) Google Scholar, Dias et al., 2004Dias J.M. Alves T. Bonifacio C. Pereira A.S. Trincao J. Bourgeois D. Moura I. Romao M.J. Structure. 2004; 12: 961-973Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). The rearrangement of this series of flexible loops revealing the H2O2 binding site only in the presence of an electron provides an elegant solution to the problem of the enzyme starting to turn over the substrate in the absence of enough reducing power, averting the risk of producing highly damaging hydroxyl radical species. With this active structure revealed, it would be intriguing to see the structural work extended around the catalytic cycle to compound I, after peroxide has bound, and to compound II, with the potential for further insights into the catalytic machinery of the peroxidase, and the mechanism of transport of electrons received by the E site heme from small redox proteins such as cytochrome c550 to the iron in the P site. Binding Dynamics of Isolated Nucleoporin Repeat Regions to Importin-βIsgro et al.StructureDecember, 2005In BriefThe nuclear pore complex, through the interaction of its proteins with transport receptors, controls the transport of large molecules into and out of the cell's nucleus. There is ample evidence for proteins with FG sequence repeats playing an essential role in this control. Previous studies have elucidated binding spots for FG sequence repeats on the surface of the transport receptor importin-β by X-ray crystallography and mutational studies. Molecular dynamics simulations have been performed to characterize the interaction of FG sequence repeats with the transport receptor. Full-Text PDF Open Archive
Referência(s)