Effects of interface mutations on association modes and electron-transfer rates between proteins

Kang, Seong A.; Crane, Brian R.

doi:10.1073/pnas.0505176102

Cited by 46 publications

(113 citation statements)

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“…It is unlikely that the effect is due to direct electrostatic repulsion of cytochrome c bound at the Pelletier/Kraut site (9). The D37K mutation could alter the conformation of CcP near Asp-34 and Asp-35, indirectly weakening the binding of cytochrome c, or Asp-37 could be part of a second binding site of the left-hand side of CcP, not yet observed in any of the crystal structures of the various cytochrome c/CcP complexes (9,47,48).…”

Section: Discussionmentioning

confidence: 99%

“…These complexes include the yeast and horse cytochrome complexes with ZnCcP (47) and four Phe-82 mutants of yeast cytochrome c binding to ZnCcP (48). In the crystal structures of all eight complexes published to date, cytochrome c binds to the same general surface region of CcP but with some variation as originally found for the yeast and horse cytochrome/CcP structures (9,48).…”

Section: Relation To Other Studiesmentioning

confidence: 94%

“…Another important aspect of the work of Crane and colleagues is that they have provided crystal structures for six additional 1:1 complexes of various modified forms of CcP and cytochrome c (47,48) to go along with the original yeast and horse cytochrome complexes with wild-type CcP provided by Pelletier and Kraut (9). These complexes include the yeast and horse cytochrome complexes with ZnCcP (47) and four Phe-82 mutants of yeast cytochrome c binding to ZnCcP (48).…”

Section: Relation To Other Studiesmentioning

confidence: 99%

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Effect of Single-Site Charge-Reversal Mutations on the Catalytic Properties of Yeast Cytochrome c Peroxidase: Mutations near the High-Affinity Cytochrome c Binding Site

et al. 2007

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Fifteen single-site charge-reversal mutations of yeast cytochrome c peroxidase (CcP) have been constructed in order to determine the effect of localized charge on the catalytic properties of the enzyme. The mutations are located on the front face of CcP, near the cytochrome c binding site identified in the crystallographic structure of the yeast cytochrome c/CcP complex (Pelletier and Kraut (1992) Science 258, 1748-1755. The mutants are characterized by absorption spectroscopy and hydrogen peroxide reactivity at both pH 6.0 and 7.5 and by steady-state kinetic studies using recombinant yeast iso-1 ferrocytochrome c(C102T) as substrate at pH 7.5. Some of the chargereversal mutations cause detectable changes in the absorption spectrum, especially at pH 7.5, reflecting changes in the equilibrium between penta-and hexa-coordinate heme species in the enzyme. An increase in the amount of hexa-coordinate heme in the mutant enzymes correlates with an increase in the fraction of enzyme that does not react with hydrogen peroxide. Steady-state velocity measurements indicate that five of the fifteen mutations cause large increases in the Michaelis constant (R31E, D34K, D37K, E118K, and E290K). These data support the hypothesis that the cytochrome c/CcP complex observed in the crystal is the dominant catalytically active complex in solution.Cytochrome c peroxidase, CcP 1 , is a detoxification enzyme localized between the inner and outer membranes of yeast mitochondria (1). CcP decreases toxic levels of hydrogen peroxide by catalyzing its reduction to water using ferrocytochrome c (2). The catalytic mechanism involves oxidation of the native enzyme by hydrogen peroxide to an enzyme intermediate called CcP Compound I, CcP-I. CcP-I contains two oxidized sites, an oxyferryl Fe(IV) heme group and a tryptophan π-cation radical located within van der Waals distance of the heme. Interaction with, and electron transfer from, ferrocytochrome c reduces CcP-I back to the native state via a second enzyme intermediate, CcP Compound II, CcP-II, completing the catalytic cycle. Since 1980, when the three-dimensional structure of CcP was first reported (3,4), CcP has played an important role in elucidating the structural basis for heme protein reactivity, † This work was supported in part by a grant from the National Institutes of Health (R15 GM59740). * Corresponding author. Phone: (815) . E-mail: jerman@niu.edu. 1 Abbreviations: Mutations in the amino acid sequences of either CcP or cytochrome c are indicated by using the one letter code for the amino acid residue in the wild-type protein, followed by the residue number and the one letter code for the amino acid residue in the mutant protein, i.e., C102T represents a mutant in which a threonine residue replaces the cysteine residue at position 102 of the wildtype protein; CcP, generic abbreviation for cytochrome c peroxidase whatever the source; yCcP, authentic yeast cytochrome c peroxidase isolated from bakers' yeast, Saccharomyces cervisiae; rCcP, recombinant CcP expressed in E. ...

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Section: Discussionmentioning

confidence: 99%

Section: Relation To Other Studiesmentioning

confidence: 94%

Section: Relation To Other Studiesmentioning

confidence: 99%

See 1 more Smart Citation

Effect of Single-Site Charge-Reversal Mutations on the Catalytic Properties of Yeast Cytochrome c Peroxidase: Mutations near the High-Affinity Cytochrome c Binding Site

et al. 2007

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“…This distance is at the long end of the range sufficient for electron transfer (32). Domain interactions in electron transfer proteins are also known to be dynamic, with a variety of donor-acceptor positions being sampled in unit time (33,34). Apparently, the nNOS hinge enables a more probable and/or efficient domain-domain interaction for the heme reduction step than does the eNOS hinge.…”

Section: Discussionmentioning

confidence: 99%

A connecting hinge represses the activity of endothelial nitric oxide synthase

Haque

Panda

Tejero

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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In mammals, endothelial nitric oxide synthase (eNOS) has the weakest activity, being one-tenth and one-sixth as active as the inducible NOS (iNOS) and the neuronal NOS (nNOS), respectively. The basis for this weak activity is unclear. We hypothesized that a hinge element that connects the FMN module in the reductase domain but is shorter and of unique composition in eNOS may be involved. To test this hypothesis, we generated an eNOS chimera that contained the nNOS hinge and two mutants that either eliminated (P728IeNOS) or incorporated (I958PnNOS) a proline residue unique to the eNOS hinge. Incorporating the nNOS hinge into eNOS increased NO synthesis activity 4-fold, to an activity two-thirds that of nNOS. It also decreased uncoupled NADPH oxidation, increased the apparent K mO2 for NO synthesis, and caused a faster heme reduction. Eliminating the hinge proline had similar, but lesser, effects. Our findings reveal that the hinge is an important regulator and show that differences in its composition restrict the activity of eNOS relative to other NOS enzymes.electron flux ͉ heme reduction ͉ kox N itric oxide (NO) is a widespread signal molecule in biology (1, 2). Three nitric oxide synthases (NOSs) generate NO in mammals [inducible NOS (iNOS), neuronal NOS (nNOS), and endothelial NOS (eNOS)]. All three are comprised of an Nterminal heme-containing oxygenase domain, an intervening calmodulin (CaM) binding sequence, and a C-terminal reductase domain that contains FMN and FAD (3). The three NOS enzymes have different gene expression patterns, protein interactions, posttranslational modifications, and catalytic behaviors that enable specific roles in biology (4-8). Of note, the NO synthesis activity of eNOS is one-10th that of iNOS and one-sixth that of nNOS. This activity is associated with a slower heme reduction in eNOS (9, 10). Which protein features enable these differences, and why they evolved, are still unclear.Studies of eNOS and nNOS chimeras established that their NO synthesis activities and heme reduction rates are primarily a function of the reductase domain (9, 11). For example, a chimera comprised of an eNOS oxygenase domain fused to an nNOS reductase domain had an NO synthesis activity and heme reduction rate that were similar to those of wild-type nNOS. This result implies that protein structural elements within the reductase domain are largely responsible for the different catalytic behaviors of eNOS and nNOS. While addressing this issue, we became interested in two hinge elements that connect the FMN subdomain of NOS to the rest of the enzyme (Fig. 1A). During catalysis, these hinge elements position the FMN subdomain to receive electrons from the NADPH-FAD module, and then may guide its interactions with the NOS oxygenase domain for electron transfer to the heme (12-17). In this way, the hinge elements could determine the rate of heme reduction and NO synthesis activity.The hinge that connects the FMN subdomain to the rest of the nNOS reductase domain is visible in the reductase crystal structure...

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“…According to several studies, the complex is dynamic, and the crystal structure might represent only a subpopulation of protein orientations (9)(10)(11)(12)(13)(14)(15). Recent studies show that the photoinduced ET between Znsubstituted CcP and Cc, both in the crystal (8,16) and in solution (17), occurs with faster backward than forward rates, indicating that the complex is present in multiple forms, only a few of which are ET-active (17).…”

mentioning

confidence: 99%

Solution structure and dynamics of the complex between cytochromecand cytochromecperoxidase determined by paramagnetic NMR

Volkov

Worrall

Holtzmann

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

244

361

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The physiological complex of yeast cytochrome c peroxidase and iso-1-cytochrome c is a paradigm for biological electron transfer. Using paramagnetic NMR spectroscopy, we have determined the conformation of the protein complex in solution, which is shown to be very similar to that observed in the crystal structure [ electron transfer ͉ encounter state ͉ transient complex ͉ spin label ͉ paramagnetic relaxation enhancement T he process of protein complex formation can be described by a two-step model, in which a short-lived, dynamic encounter complex precedes a dominant, well defined state (Fig. 1). The former enables proteins to undergo reduced-dimensionality search of the optimal binding geometry, thereby accelerating molecular association as compared with 3D diffusion (1). Fast molecular association is essential for protein-protein complexes that require high turnover rates, like those involved in electron transfer (ET) in photosynthesis, respiration, and other metabolic processes (2). The physiological complex of yeast iso-1-cytochrome c (Cc) and yeast cytochrome c peroxidase (CcP) is a paradigm for the intermolecular ET (3) and is one of the few transient ET complexes for which a crystal structure has been solved (4). A recent study has confirmed that the protein-protein orientation observed in the crystal is ET-active (5). However, it has remained a matter of debate whether this structure represents the only form in solution (6-8). According to several studies, the complex is dynamic, and the crystal structure might represent only a subpopulation of protein orientations (9-15). Recent studies show that the photoinduced ET between Znsubstituted CcP and Cc, both in the crystal (8, 16) and in solution (17), occurs with faster backward than forward rates, indicating that the complex is present in multiple forms, only a few of which are .Characterization of the binding interface in the dynamic encounter state (Fig. 1B) has so far proven to be elusive. Investigation of protein complexes by using x-ray crystallography or conventional NMR spectroscopy addresses only the singleorientation species (Fig. 1C), and the only way to visualize the dynamic state is offered by theoretical modeling studies (11,18). In the recent, elegant work of Clore and coworkers (19,20), it was shown how paramagnetic relaxation can be applied to study the dynamic state of protein-DNA complexes. We report on the application of an analogous experimental approach that allows us to define both the dominant protein-protein orientation (Fig. 1C) and the conformational space sampled by the proteins in the dynamic encounter complex (Fig. 1B). Results and DiscussionSolution Structure of the Complex. The concept of the approach is that NMR resonance intensities of one of the proteins in the complex are affected by a paramagnetic spin label covalently attached to the other protein (Fig. 2). The paramagnetic effects are converted into distance restraints (21-23), which can be used to calculate protein-protein orientations within the complex (24). Five ...

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Effects of interface mutations on association modes and electron-transfer rates between proteins

Cited by 46 publications

References 49 publications

Effect of Single-Site Charge-Reversal Mutations on the Catalytic Properties of Yeast Cytochrome c Peroxidase: Mutations near the High-Affinity Cytochrome c Binding Site

Effect of Single-Site Charge-Reversal Mutations on the Catalytic Properties of Yeast Cytochrome c Peroxidase: Mutations near the High-Affinity Cytochrome c Binding Site

A connecting hinge represses the activity of endothelial nitric oxide synthase

Solution structure and dynamics of the complex between cytochromecand cytochromecperoxidase determined by paramagnetic NMR

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