Crystal structure of nitric oxide inhibited cytochrome c peroxidase

Edwards, Sarah; Kraut, Joseph; Poulos, T.L.

doi:10.1021/bi00421a016

Cited by 36 publications

(27 citation statements)

References 37 publications

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“…Although the details of how electrons are transferred from reduced cyt c to CCP are still unclear, the crystal structure of the CCP-cytochrome c complex does indicate that cyt c binds near the loop 190-195 region of CCP (Pelletier & Kraut, 1992). The significantly increased thermodynamic motion of loop 190-195 was not only found in the F202G mutant protein, but also in a previously reported structure of nitric oxide (NO) bound to the heme Fe of wtCPP, where a slight movement of TrpI9' away from His"' and an increase in the B-factor of TrpI9' from 18.7 A2 to 28.3 A2 was observed (Edwards et al, 1988;Edwards & Poulos, 1990). This experiment reveals that the binding of NO to the distal side of the heme iron, the same site where peroxide binds, can specifically affect the thermodynamic motion of loop [190][191][192][193][194][195].…”

Section: Is the Loop Opening Involved In The Electron Transfer Process?mentioning

confidence: 51%

Protein conformer selection by ligand binding observed with crystallography

et al. 1998

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A large-scale movement between "closed' and "open" conformations of a protein loop was observed directly with protein crystallography by trapping individual conformers through binding of an exogenous ligand and characterization with solution kinetics. The buried indole ring of TrpI9' in cytochrome c peroxidase (CCP) was displaced by exogenous ligands, causing a conformational change of loop P r~'~~-A s n '~~ and exposing TrpI9' to the protein surface. Kinetic measurements are consistent with a two-step binding mechanism in which the rate-limiting step is a transition of the protein to the open state, which then binds the ligand. This large-scale conformational change of a functionally important region of CCP is independent of ligand and indicates that about 4% of the wild-type protein is in the open form in solution at any given time.Keywords: artificial cavity; cytochrome c peroxidase; kinetics; ligand binding; loop movement; protein crystallography; site-directed mutagenesis Much evidence exists that proteins exhibit considerable flexibility. Ligands are often found in the center of proteins with no observable path to the surface, suggesting that the protein must transiently unfold in order for the ligand to enter. Examples include myoglobin and hemoglobin oxygen binding to the heme (Karplus & Petsko, 1990), fatty acid binding to fatty acid binding protein (LaLonde et al., 1994), retinol binding to retinol binding protein (Zanotti et al., 1993), and ligand binding to artificial cavities introduced into proteins (Eriksson et al., 1992;Fitzgerald et al., 1994Fitzgerald et al., , 1996Feher et al., 1996). Flexible surface-loops in protein structure are thought to play an important role in substrate binding and molecular recognition. Crystal structures have shown that, for a number of enzymes and antibodies, substrate and/or antigen binding causes a conformational change of a surface-loop either from an "open" to a "closed" form to partially cover the bound substrate (Joseph et al., 1990;Lolis & Petsko, 1990;Derewenda et al., 1992;Jia et al., 1995), or from a "partially closed' to "open" form, resulting in an induced fit for the antigen or ligand binding (Rini et al., 1992;Concha et al., 1993). These conformational changes have been observed directly by NMR, and the relevance of these motions to dihydrofolate reductase function has been demonstrated (Epstein et al., 1995). All of these examples reveal that Reprint requests to: Duncan E. McRee, Department of Molecular Biology"MB8, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, California 92037; e-mail: dem@scripps.edu.Abbreviations: CCP, cytochrome c peroxidase; wtCCP, wild-type CCP; cyt c, cytochrome c; DMI', 1,2-dimethylimidazolium; 2a5mT+, 2-amino-5-methylthiazolium; MPD, 2-methyl-2,4-pentanediol.proteins switch between conformations and that these changes are often key to protein function.Here we trap a large conformational change of a protein loop by binding an exogenous ligand to the protein and directly visualizing the structure...

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Section: Is the Loop Opening Involved In The Electron Transfer Process?mentioning

confidence: 51%

Protein conformer selection by ligand binding observed with crystallography

et al. 1998

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“…1% of the total reduced N oxides), and the proportion of NzO product is not particularly sensitive to addition of excess NO or to its removal by sparging. Instead, addition of NO results only in significant inhibition of NO production, consistent with the formation of stable ferroheme protein-NO complexes [34,35]. There is no evidence that NO interacts with the heme cd, enzyme in a productive fashion to enhance N,O production.…”

Section: Effect Of No On Cu Nir Activitymentioning

confidence: 80%

Evidence for an NO‐rebound, mechanism for production of N₂O from nitrite by the copper‐containing nitrite reductase from Achromobacter cycloclastes

1991

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Reduction of NO; by the C&containing nitrite reductase from Achromobacfer cycloclustes produces NO as the primary product initially, but as NO accumulates, NO production levels-off and N,O production becomes significant. Reaction of the enzyme with NO; in the presence of NO increases the amount of NzO product significantly, while trapping the NO product as nitrosylhemoglobin or rapid. removal of NO by spaiging results in no detectable N20 production. Reaction of the enzyme with "NO; in the presence of 14N0 results in rapid formation of the mixed isotope product (idId, "N)O in ca. 45% yield. In contrast, the presence or absence of NO has no effect on N,O production by a prototypical heme &,-containing nitrite reductase. These results are consistent with formation of a labile Cu'-NO" species in the copper enzyme, which normally decomposes to NO. Production of N,O requires that the released NO must rebind to the enzyme to combine with a second NO; or a species derived therefrom.Denitrification; Nitrite reductase; Nitrosyl; Isotope labelling; Reaction mechanism; N1O production INTRODUCTIONDespite many detailed studies on intact bacteria, crude extracts, and purified enzymes [l-4], the mechanism of denitrification continues to be the object of controversy. Although it is now generally accepted that denitrifying bacteria possess a distinct nitric oxide reductase (NOR) activity and that at least a significant portion of the total nitrogen flux occurs via a stepwise pathway with NO as an intermediate (Eqn, 1), the importance of an alternative direct pathway in which 2 nitrite ions are reduced to N20 on a single enzyme, nitrite reductase (NiR), (Eqn. 2) remains unclear.On the one hand, quantitative studies of NO levels during denitrification by several organisms have been interpreted as indicating that only the stepwise pathway of Eqn. (1) NO [12], indicating that, for some organisms at least, NO is not an obligatory intermediate. This conclusion is supported by a number of isotope labelling and trapping studies [ 13-161, by the fact that the "N isotope effect increases with increasing nitrite concentration, suggesting that 2 nitrite ions combine prior to the first irreversible step [ 17,181, and by the fact that purified NiR's do in at least some cases produce significant amounts of N20 that cannot be attributed to chemical side reactions [19-211. In order to determine the origin of some of these apparently contradictory findings, we have examined the reduction of NO, by both the Cu-and heme cd,-containing NiR's in some detail. The problem is complicated by the fact that one of the products, NO, is known to be a potent inhibitor of NO; reduction by both types of enzyme [22,23]. In this communication, WC prcscnt evidence that NO binding to the Cu NiR of Volume 291, number 1 FEBS LETTERSOctober 199 1Achrornobacter cycioclastes is essential for N20 production by the enzyme. r EXPERIMENTALNiteite reductases were isolated from A. cyclocl~srcs [24] and Pseudomonos stutzeri [ZS] as peeviously described and stored frozen un...

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“…7) and presence ( Fig. 9) of Cl movement of a number of amino acid residues and a rearrangement of active site water molecules) have been described for cytochrome c peroxidase (26). Moreover, slower rates of NO binding to the Fe(III) forms of a number of heme proteins have been attributed to ligand replacement (21,67).…”

Section: •-mentioning

confidence: 89%

“…Likewise, a variety of studies have documented the ability of NO to bind to the heme moiety of peroxidases (24)(25)(26)(27). Both spectroscopic and rapid kinetics measurements were recently used to demonstrate that NO rapidly binds to both ferric [Fe(III)] and ferrous [Fe(II)] forms of myeloperoxidase (MPO) (28), a hemoprotein which is present in abundance in neutrophils, monocytes and certain sub-populations of tissue macrophages, such as in atherosclerotic lesions (29,30).…”

Section: •-mentioning

confidence: 99%