Multinuclear magnetic resonance studies of the 2Fe.2S* ferredoxin from Anabaena species strain PCC 7120. 2. Sequence-specific carbon-13 and nitrogen-15 resonance assignments of the oxidized form

Multinuclear two-dimensional NMR techniques were used to assign nearly all diamagnetic 13C and 15N resonances of the plant-type 2Fe.2S* ferredoxin from Anabaena sp. strain PCC 7120. Since a 13C spin system directed strategy had been used to identify the 1H spin systems [Oh, B.-H., Westler, W. M., & Markley, J. L. (1989) J. Am. Chem. Soc. 111, 3083-3085], the sequence-specific 1H assignments [Oh, B.-H., & Markley, J. L. (1990) Biochemistry (first paper of three in this issue)] also provided sequence-specific 13C assignments. Several resonances from 1H-13C groups were assigned independently of the 1H assignments by considering the distances between these nuclei and the paramagnetic 2Fe.2S* center. A 13C-15N correlation data set was used to assign additional carbonyl carbons and to analyze overlapping regions of the 13C-13C correlation spectrum. Sequence-specific assignments of backbone and side-chain nitrogens were based on 1H-15N and 13C-15N correlations obtained from various two-dimensional NMR experiments.     

NMR spectroscopic studies of the hydrogenosomal [2Fe-2S] ferredoxin from Trichomonas vaginalis: hyperfine-shifted 1H resonances

The hyperfine-shifted 1H NMR resonances of oxidized and reduced Trichomonas vaginalis ferredoxin, a functionally unique [2Fe-2S] ferredoxin, have been studied. The oxidized protein spectrum displayed a pattern of six broad upfield-shifted resonances between 13 and 40 ppm with chemical shifts distinct from those of other [2Fe-2S] ferredoxins. All hyperfine 1H resonances of the oxidized ferredoxin displayed anti-Curie temperature dependences. Reduced T. vaginalis ferredoxin displayed hyperfine resonances both upfield and downfield of the diamagnetic region. These resonances showed Curie temperature dependences. Overall the hyperfine-shifted NMR spectrum of T. vaginalis ferredoxin, along with other spectroscopic properties, suggested different structural properties for the active center of oxidized hydrogenosomal ferredoxins from those of other [2Fe-2S] ferredoxins.     

Assignment of 1H and 13C hyperfine-shifted resonances for tuna ferricytochrome c.

Tuna ferricytochrome c has been used to demonstrate the potential for completely assigning 1H and 13C strongly hyperfine-shifted resonances in metalloprotein paramagnetic centers. This was done by implementation of standard two-dimensional NMR experiments adapted to take advantage of the enhanced relaxation rates of strongly hyperfine-shifted nuclei. The results show that complete proton assignments of the heme and axial ligands can be achieved, and that assignments of several strongly shifted protons from amino acids located close to the heme can also be made. Virtually all proton-bearing heme 13C resonances have been located, and additional 13C resonances from heme vicinity amino acids are also identified. These results represent an improvement over previous proton resonance assignment efforts that were predicated on the knowledge of specific assignments in the diamagnetic protein and relied on magnetization transfer experiments in heterogeneous solutions composed of mixtures of diamagnetic ferrocytochrome c and paramagnetic ferricytochrome c. Even with that more complicated procedure, complete heme proton assignments for ferricytochrome c have never been demonstrated by a single laboratory. The results presented here were achieved using a more generally applicable strategy with a solution of the uniformly oxidized protein, thereby eliminating the requirement of fast electron self-exchange, which is a condition that is frequently not met.     

Multinuclear magnetic resonance studies of the 2Fe.2S* ferredoxin from Anabaena species strain PCC 7120. 3. Detection and characterization of hyperfine-shifted nitrogen-15 and hydrogen-1 resonances of the oxidized form

All the nitrogen signals from the amino acid side chains and 80 of the total of 98 backbone nitrogen signals of the oxidized form of the 2Fe.2S* ferredoxin from Anabaena sp. strain PCC 7120 were assigned by means of a series of heteronuclear two-dimensional experiments [Oh, B.-H. Mooberry, E. S., & Markley, J. L. (1990) Biochemistry (second paper of three in this issue )]. Two additional nitrogen signals were observed in the one-dimensional 15N NMR spectrum and classified as backbone amide resonances from residues whose proton resonances experience paramagnetic broadening. The one-dimensional 15N NMR spectrum shows nine resonances that are hyperfine shifted and broadened. From this inventory of diamagnetic nitrogen signals and the available X-ray coordinates of a related ferredoxin [Tsukihara, T., Fukuyama, K., Nakamura, M., Katsube, Y., Tanaka, N., Kakudo, M., Wada, K., Hase, T., & Matsubara, H. (1981) J. Biochem. 90, 1763-1773], the resolved hyperfine-shifted 15N peaks were attributed to backbone amide nitrogens of the nine amino acids that share electrons with the 2Fe.2S* center or to backbone amide nitrogens of two other amino acids that are close to the 2Fe.2S* center. The seven 15N signals that are missing and unaccounted for probably are buried under the envelope of amide signals. 1H NMR signals from all the amide protons directly bonded to the seven missing and nine hyperfine-shifted nitrogens were too broad to be resolved in conventional 2D NMR spectra.(ABSTRACT TRUNCATED AT 250 WORDS)     

Multinuclear magnetic resonance studies of the 2Fe.2S* ferredoxin from Anabaena species strain PCC 7120. 1. Sequence-specific hydrogen-1 resonance assignments and secondary structure in solution of the oxidized form

Complete sequence-specific assignments were determined for the diamagnetic 1H resonances from Anabaena 7120 ferredoxin (Mr = 11,000). A novel assignment procedure was followed whose first step was the identification of the 13C spin systems of the amino acids by a 13C(13C) double quantum correlation experiment [Oh, B.-H., Westler, M. W., Darba, P., & Markley, J. L. (1988) Science 240, 908-911]. Then, the 1H spin systems of the amino acids were identified from the 13C spin systems by means of direct and relayed 1H(13C) single-bond correlations [Oh, B.-H., Westler, W. M., & Markley, J. L. (1989) J. Am. Chem. Soc. 111, 3083-3085]. The sequential resonance assignments were based mainly on conventional interresidue 1H alpha i-1HNi + 1 NOE connectivities. Resonances from 18 residues were not resolved in two-dimensional 1H NMR spectra. When these residues were mapped onto the X-ray crystal structure of the homologous ferredoxin from Spirulina platensis [Fukuyama, K., Hase, T., Matsumoto, S., Tsukihara, T., Katsube, Y., Tanaka, N., Kakudo, M., Wada, K., & Matsubara, H. (1980) Nature 286, 522-524], it was found that they correspond to amino acids close to the paramagnetic 2Fe.2S* cluster. Cross peaks in two-dimensional homonuclear 1H NMR spectra were not observed for any protons closer than about 7.8 A to both iron atoms. Secondary structural features identified in solution include two antiparallel beta-sheets, one parallel beta-sheet, and one alpha-helix.     

Sequential resonance assignments of oxidized high-potential iron-sulfur protein from Chromatium vinosum.

2D NMR spectra of the high-potential iron-sulfur protein (HiPIP) from Chromatium vinosum have been used to obtain partial resonance assignments for the oxidized paramagnetic redox state of the protein. Sequence-specific assignments were made using NOESY and COSY spectra in H2O and D2O of the following backbone segments: Asn-5-Arg-33, Glu-39-Asp-45, Gly-55-Cys-63, Gly-68-Ala-78, and Leu-82-Gly-85. NOESY spectra with a spectral width wide enough to include the hyperfine-shifted resonances revealed numerous NOE contacts between these signals and those in the main envelope of the proton spectrum. With the aid of the X-ray crystal structure [Carter, C.W., Kraut, J., Freer, S. T., Xuong, N. H., Alden, R. A., & Bartsch, R. G. (1974) J. Biol. Chem. 249, 4212], these NOEs permitted seven of the nine hyperfine-shifted signals to be assigned to three of the cysteine residues liganded to the metal cluster (Cys-43, Cys-46, and Cys-77). The other two hyperfine-shifted signals produced no detectable NOEs to other resonances in the spectrum and were tentatively assigned to the remaining cysteinyl ligand (Cys-63). These assignments, in conjunction with recent theoretical models of the electronic structure of the Fe4S4 cluster [Noodleman, L. (1988) Inorg. Chem. 27, 3677; Bertini, I., Briganti, F., Luchinat, C., Scozzafava, A., & Sola, M. (1991) J. Am. Chem. Soc. 113, 1237], indicate that the iron atoms coordinated to Cys-63 and Cys-77 are those of the mixed-valence Fe(3+)-Fe2+ pair whereas Cys-43 and Cys-46 are ligands to the Fe(3+)-Fe3+ metal pair.     

Acceleration of protein backbone NMR assignment by combinatorial labeling: Application to a small molecule binding study

Selective labeling with stable isotopes has long been recognized as a valuable tool in protein NMR to alleviate signal overlap and sensitivity limitations. In this study, combinatorial ¹⁵N‐, ¹³C^α‐, and ¹³C'‐selective labeling has been used during the backbone assignment of human cyclophilin D to explore binding of an inhibitor molecule. Using a cell‐free expression system, a scheme that involves ¹⁵N, 1‐¹³C, 2‐¹³C, fully ¹⁵N/¹³C, and unlabeled amino acids was optimized to gain a maximum of assignment information from three samples. This scheme was combined with time‐shared triple‐resonance NMR experiments, which allows a fast and efficient backbone assignment by giving the unambiguous assignment of unique amino acid pairs in the protein, the identity of ambiguous pairs and information about all 19 non‐proline amino acid types. It is therefore well suited for binding studies where de novo assignments of amide ¹H and ¹⁵N resonances need to be obtained, even in cases where sensitivity is the limiting factor.     

Assignments of backbone 1H, 13C, and 15N resonances and secondary structure of ribonuclease H from Escherichia coli by heteronuclear three-dimensional NMR spectroscopy.

The assignments of individual magnetic resonances of backbone nuclei of a larger protein, ribonuclease H from Escherichia coli, which consists of 155 amino acid residues and has a molecular mass of 17.6 kDa are presented. To remove the problem of degenerate chemical shifts, which is inevitable in proteins of this size, three-dimensional NMR was applied. The strategy for the sequential assignment was, first, resonance peaks of amides were classified into 15 amino acid types by 1H-15N HMQC experiments with samples in which specific amino acids were labeled with 15N. Second, the amide 1H-15N peaks were connected along the amino acid sequence by tracing intraresidue and sequential NOE cross peaks. In order to obtain unambiguous NOE connectivities, four types of heteronuclear 3D NMR techniques, 1H-15N-1H 3D NOESY-HMQC, 1H-15N-1H 3D TOCSY-HMQC, 13C-1H-1H 3D HMQC-NOESY, and 13C-1H-1H 3D HMQC-TOCSY, were applied to proteins uniformly labeled either with 15N or with 13C. This method gave a systematic way to assign backbone nuclei (N, NH, C alpha H, and C alpha) of larger proteins. Results of the sequential assignments and identification of secondary structure elements that were revealed by NOE cross peaks among backbone protons are reported.     

1H NMR spectra of vertebrate [2Fe-2S] ferredoxins. Hyperfine resonances suggest different electron delocalization patterns from plant ferredoxins

We report the observation of paramagnetically shifted (hyperfine) proton resonances from vertebrate mitochondrial [2Fe-2S] ferredoxins. The hyperfine signals of human, bovine, and chick [2Fe-2S] ferredoxins are described and compared with those of Anabaena 7120 vegetative ferredoxin, a plant-type [2Fe-2S] ferredoxin studied previously [Skjeldal, L., Westler, W. M., & Markley, J. L. (1990) Arch. Biochem. Biophys. 278, 482-485]. The hyperfine resonances of the three vertebrate ferredoxins were very similar to one another both in the oxidized state and in the reduced state, and slow (on the NMR scale) electron self-exchange was observed in partially reduced samples. For the oxidized vertebrate ferredoxins, hyperfine signals were observed downfield of the diamagnetic envelope from +13 to +50 ppm, and the general pattern of peaks and their anti-Curie temperature dependence are similar to those observed for the oxidized plant-type ferredoxins. For the reduced vertebrate ferredoxins, hyperfine signals were observed both upfield (-2 to -18 ppm) and downfield (+15 to +45 ppm), and all were found to exhibit Curie-type temperature dependence. This pattern and temperature dependence are distinctly different from those found with reduced plant-type ferredoxins which have signal centered around +120 ppm with Curie-type temperature dependence, assigned to cysteines which interact with Fe(III), and signals centered around +20 ppm with anti-Curie temperature dependence, assigned to cysteines which interact with Fe(II) [Dugad, L. B., La Mar, G. N., Banci, L., & Bertini, I. (1990) Biochemistry 29, 2263-2271].(ABSTRACT TRUNCATED AT 250 WORDS)     

Assignment of paramagnetically shifted resonances in the 1H NMR spectrum of horse ferricytochrome c.

The proton resonances of the heme, the axial ligands, and other hyperfine-shifted resonances in the 1H nuclear magnetic resonance spectrum of horse ferricytochrome c have been investigated by means of one- and two-dimensional nuclear Overhauser and magnetization transfer methods. Conditions for saturation transfer experiments in mixtures of ferro- and ferricytochrome c were optimized for the cross assignment of corresponding resonances in the two oxidation states. New resonance assignments were obtained for the methine protons of both thioether bridges, the beta and gamma meso protons, the propionate six heme substituent, the N pi H of His-18, and the Tyr-67 OH. In addition, several recently reported assignments were confirmed. All of the resolved hyperfine-shifted resonances in the spectrum of ferricytochrome c are now identified. The Fermi contact shifts experienced by the heme and ligand protons are discussed.     

Structure-function relationship of [2Fe2S] ferredoxins and design of a model molecule

[2Fe2S] ferredoxins isolated from various plants and algae comprise 93–99 amino acid residues and resemble each other not only in sequences, but also in physiological functions. One of them isolated from Spirulina platensis was subjected to X-ray analysis and its three dimensional structure is now known. [2Fe2S] ferredoxins of a different type are found in halobacteria and comprise 128 amino acid residues. Both types of the [2Fe2S] ferredoxins exhibit low redox potentials. By comparing the amino acid sequences of 28 [2Fe2S] ferredoxins and the tertiary structure of S. platensis ferredoxin we predicted a common three-dimensional structure to the [2Fe2S] ferredoxins and proposed a molecular surface area to be interacting with FNR. An artificial small molecule composed of 20 amino acid residues is designed on the basis of the tertiary structure of S. platensis ferredoxin. The amino acid sequence was predicted to be ProTyrSerCysArgAlaGlyAlaCysSerThrCysAlaGly ProLeuLeuThr CysVal which should have a [2Fe2S] cluster with a low redox potential     

Resonance assignment of 13C/15N labeled solid proteins by two- and three-dimensional magic-angle-spinning NMR

The comprehensive structure determination of isotopically labeled proteins by solid-state NMR requires sequence-specific assignment of ¹³C and ¹⁵ N spectra. We describe several 2D and 3D MAS correlation techniques for resonance assignment and apply them, at 7.0 Tesla, to ¹³C and ¹⁵N labeled ubiquitin to examine the extent of resonance assignments in the solid state. Both interresidue and intraresidue assignments of the ¹³C and ¹⁵N resonances are addressed. The interresidue assignment was carried out by an N(CO)CA technique, which yields N_i-C_i–1 connectivities in protein backbones via two steps of dipolar-mediated coherence transfer. The intraresidue connectivities were obtained from a new 3D NCACB technique, which utilizes the well resolved C chemical shift to distinguish the different amino acids. Additional amino acid type assignment was provided by a ¹³C spin diffusion experiment, which exhibits ¹³C spin pairs as off-diagonal intensities in the 2D spectrum. To better resolve carbons with similar chemical shifts, we also performed a dipolar-mediated INADEQUATE experiment. By cross-referencing these spectra and exploiting the selective and extensive ¹³ C labeling approach, we assigned 25% of the amino acids in ubiquitin sequence-specifically and 47% of the residues to the amino acid types. The sensitivity and resolution of these experiments are evaluated, especially in the context of the selective and extensive ¹³C labeling approach.     

Amino acid selective unlabeling for sequence specific resonance assignments in proteins

Sequence specific resonance assignment constitutes an important step towards high-resolution structure determination of proteins by NMR and is aided by selective identification and assignment of amino acid types. The traditional approach to selective labeling yields only the chemical shifts of the particular amino acid being selected and does not help in establishing a link between adjacent residues along the polypeptide chain, which is important for sequential assignments. An alternative approach is the method of amino acid selective ‘unlabeling’ or reverse labeling, which involves selective unlabeling of specific amino acid types against a uniformly ¹³C/¹⁵N labeled background. Based on this method, we present a novel approach for sequential assignments in proteins. The method involves a new NMR experiment named, {¹²CO _i –¹⁵N _i+1}-filtered HSQC, which aids in linking the ¹H^N/¹⁵N resonances of the selectively unlabeled residue, i, and its C-terminal neighbor, i + 1, in HN-detected double and triple resonance spectra. This leads to the assignment of a tri-peptide segment from the knowledge of the amino acid types of residues: i − 1, i and i + 1, thereby speeding up the sequential assignment process. The method has the advantage of being relatively inexpensive, applicable to ²H labeled protein and can be coupled with cell-free synthesis and/or automated assignment approaches. A detailed survey involving unlabeling of different amino acid types individually or in pairs reveals that the proposed approach is also robust to misincorporation of ¹⁴N at undesired sites. Taken together, this study represents the first application of selective unlabeling for sequence specific resonance assignments and opens up new avenues to using this methodology in protein structural studies.     

A general method for assigning NMR spectra of denatured proteins using 3D HC(CO)NH-TOCSY triple resonance experiments

Summary A general approach for assigning the resonances of uniformly ¹⁵N- and ¹³C-labeled proteins in their unfolded state is presented. The assignment approach takes advantage of the spectral dispersion of the amide nitrogen chemical shifts in denatured proteins by correlating side chain and backbone carbon and proton frequencies with the amide resonances of the same and adiacent residues. The ¹H resonances of the individual amino acid spin systems are correlated with their intraresidue amide in a 3D ¹⁵N-edited ¹H, ¹H-TOCSY-HSQC experiment, which allows the spin systems to be assigned to amino acid type. The spin systems are then linked to the adjacent i-1 spin system using the 3D H(C)(CO)NH-TOCSY experiment. Complete ¹³C assignments are obtained from the 3D (H)C(CO)NH-TOCSY experiment. Unlike other methods for assigning denatured proteins, this approach does not require previous knowledge of the native state assignments or specific interconversion rates between the native and denatured forms. The strategy is demonstrated by assigning the ¹H, ¹³C, and ¹⁵N resonances of the FK506 binding protein denatured in 6.3 M urea.     

A single amino acid change makes a rat neuronal sodium channel highly sensitive to pyrethroid insecticides

Vitamin K-dependent protein S, which is a cofactor for activated protein C and thus important for down-regulation of the coagulation cascade, contains several Ca(2+)-binding sites with unusually high affinity. The 89 amino acid fragment constituting the third and fourth epidermal growth factor-like (EGF) modules of protein S is the smallest fragment that retains high-affinity Ca(2+) binding and is therefore useful for investigating the structural basis of this property. Heteronuclear multidimensional nuclear magnetic resonance experiments were used to obtain extensive assignments of the (1)H, 15N and (13)C resonances of the module pair with one Ca(2+) bound in EGF 4. In addition, nearly complete assignments of the (1)H resonances of the isolated Ca(2+)-free EGF 3 module were obtained. The assignment process was complicated by broadening of several resonances, spectral heterogeneity caused by cis-trans isomerisation of the peptide bond preceding Pro-168, and dimerisation. Analysis of weighted average secondary chemical shifts, (3)J(HNHalpha) coupling constants, and NOE connectivities suggest that both EGF modules in this fragment adhere to the classical secondary structure of EGF modules, consisting of one major and one minor anti-parallel beta-sheet.     

Automated amino acid side-chain NMR assignment of proteins using <Superscript>13</Superscript>C- and <Superscript>15</Superscript>N-resolved 3D [<Superscript>1</Superscript>H,<Superscript>1</Superscript>H]-NOESY

ASCAN is a new algorithm for automatic sequence-specific NMR assignment of amino acid side-chains in proteins, which uses as input the primary structure of the protein, chemical shift lists of (1)H(N), (15)N, (13)C(alpha), (13)C(beta) and possibly (1)H(alpha) from the previous polypeptide backbone assignment, and one or several 3D (13)C- or (15)N-resolved [(1)H,(1)H]-NOESY spectra. ASCAN has also been laid out for the use of TOCSY-type data sets as supplementary input. The program assigns new resonances based on comparison of the NMR signals expected from the chemical structure with the experimentally observed NOESY peak patterns. The core parts of the algorithm are a procedure for generating expected peak positions, which is based on variable combinations of assigned and unassigned resonances that arise for the different amino acid types during the assignment procedure, and a corresponding set of acceptance criteria for assignments based on the NMR experiments used. Expected patterns of NOESY cross peaks involving unassigned resonances are generated using the list of previously assigned resonances, and tentative chemical shift values for the unassigned signals taken from the BMRB statistics for globular proteins. Use of this approach with the 101-amino acid residue protein FimD(25-125) resulted in 84% of the hydrogen atoms and their covalently bound heavy atoms being assigned with a correctness rate of 90%. Use of these side-chain assignments as input for automated NOE assignment and structure calculation with the ATNOS/CANDID/DYANA program suite yielded structure bundles of comparable quality, in terms of precision and accuracy of the atomic coordinates, as those of a reference structure determined with interactive assignment procedures. A rationale for the high quality of the ASCAN-based structure determination results from an analysis of the distribution of the assigned side chains, which revealed near-complete assignments in the core of the protein, with most of the incompletely assigned residues located at or near the protein surface.     

Specific labeling approaches to guanine and adenine imino and amino proton assignments in the AMP–RNA aptamer complex

The secondary structure of a recently identified ATP-binding RNA aptamer consists of apurine-rich 11-residue internal loop positioned opposite a single guanine bulge flanked oneither side by helical stem segments. The ATP ligand targets the internal loop and bulgedomains, inducing a structural transition in this RNA segment on complex formation.Specifically, 10 new slowly exchanging proton resonances in the imino, amino and sugarhydroxyl chemical shift range are observed on AMP–RNA aptamer complex formation.This paper outlines site-specific labeling approaches to identify slowly exchanging imino(guanine) and amino (guanine and adenine) protons in internal loop and bulge segments ofcompact RNA folds such as found in the AMP–RNA aptamer complex. One approachincorporates 15N-labeled guanine (N1 imino and N2 amino positions) and 15N-labeledadenine (N6 amino position), one residue at a time, in the AMP-binding RNA aptamer, withlabeling incorporation through chemical synthesis facilitated by generating the aptamer fromtwo separate strands. The unambiguous assignments deduced from the 15N labeling studieshave been verified from an independent labeling strategy where individual guanines in theinternal loop have been replaced, one at a time, by inosines and assignments were made onthe basis of the large 2 ppm downfield shift of the guanine imino protons on inosinesubstitution. The strengths and limitations of the inosine-for-guanine substitution approachemerge from our studies on the AMP–RNA aptamer complex. The assignment of theinternal loop and bulge imino and amino protons was critical in our efforts to define thesolution structure of the AMP–RNA aptamer complex since these slowly exchangingprotons exhibit a large number of long-range intramolecular NOEs within the RNA, as wellas intermolecular NOEs to the AMP in the complex. The current application of specific 15Nand inosine labeling approaches for exchangeable imino and amino proton assignments in thenonhelical segments of an RNA aptamer complex in our laboratory complements selective 2Hand 13C approaches to assign nonexchangeable base and sugar protons in RNA andligand–RNA complexes reported in the literature.     

Proton homonuclear correlated spectroscopy as an assignment tool for hyperfine-shifted resonances in medium-sized paramagnetic proteins: cyanide-ligated yeast cytochrome c peroxidase as an example.

Two types of homonuclear proton COSY experiments are shown to be useful in making resonance assignments in cyanide-ligated cytochrome c peroxidase, a 34 kDa paramagnetic heme protein. Both magnitude COSY and phase-sensitive COSY experiments provide spectra useful for making proton assignments to resonances of strongly relaxed hyperfine-shifted protons. This initial investigation demonstrates that COSY experiments combined with NOESY experiments are feasible for hyperfine-shifted protons of paramagnetic proteins larger than metmyoglobins and ferricytochromes c, for which the nuclear spin-lattice relaxation times are in the range 70-300 ms. Taken together, COSY and NOESY experiments, although not yet widely applied to paramagnetic metalloproteins, provide a reliable protocol for accurately assigning hyperfine-shifted resonances that are part of a metalloenzyme's active site. Specific examples of expected proton homonuclear COSY connectivities that were not observed in these experiments are presented, and utilization of COSY with respect to the proton resonance line widths and apparent nuclear relaxation times is discussed. The COSY experiments presented here provide valuable verification of previously proposed hyperfine resonance assignments for cyanide-ligated cytochrome c peroxidase, which were made by using NOESY experiments alone, and in several instances expand these assignments to additional protons in particular amino acid spin systems.     

A nuclear Overhauser effect study of the heme crevice in the resting state and compound I of horseradish peroxidase: evidence for cation radical delocalization to the proximal histidine

The assignment of resolved hyperfine-shifted resonances in high-spin resting state horseradish peroxidase (HRP) and its double-oxidized reactive form, compound I (HRP-I), has been carried out by using the nuclear Overhauser effect (NOE) starting with the known heme methyl assignments in each species. In spite of the efficient spin-lattice relaxation and very broad resonances, significant NOEs were observed for all neighboring pyrrole substituents, which allowed the assignment of the elusive propionate alpha-methylene protons. In the resting state HRP, this leads directly to the identity of the proximal His-170 H beta peaks. The determination that one of the most strongly contact-shifted single proton resonances in HRP-I does not arise from the porphyrin dictates that the cation radical must be delocalized to some amino acid residue. The relaxation properties of the non-heme contact-shifted signal in HRP-I support the identity of this contributing residue as the proximal His-170. Detailed analysis of changes in both contact shift pattern and NOEs indicates that compound I formation is accompanied by a approximately 5 degree rotation of the 6-propionate group. The implication of a porphyrin cation radical delocalized over the proximal histidine for the proposed location of the solely amino acid centered radical in compound I of related cytochrome c peroxidase is discussed.     

N-Isotope effects on the Raman spectra of Fe2S2 ferredoxin and Rieske ferredoxin: evidence for structural rigidity of metal sites

The diiron ferredoxins have a common diamond-core structure with two bridging sulfides, but differ in the nature of their terminal ligands: either four cysteine thiolates in the Fe(2)S(2) ferredoxins or two cysteine thiolates and two histidine imidazoles in the Rieske ferredoxins. Contributions of the bridging (b) and terminal (t) ligands to the resonance Raman spectra of the Fe(2)S(2) ferredoxins have been distinguished previously by isotopic substitution of the bridging sulfides. We now find that uniform (15)N-labeling of Anabaena Fe(2)S(2) ferredoxin results in shifts of -1 cm(-1) in the Fe-S(t) stretching modes at 282, 340, and 357 cm(-1). The (15)N dependence is ascribed to kinematic coupling of the Fe-S(Cys) stretch with deformations of the cysteine backbone, including the amide nitrogen. No (15)N dependence occurs for the nu(Fe-S(b)) modes at 395 and 426 cm(-1). Similar effects are observed for the Rieske center in T4MOC ferredoxin from the toluene-4-monooxygenase system of Pseudomonas mendocina. Upon selective (15)N-labeling of the alpha-amino group of cysteine, the vibrational modes at 321, 332, 350, and 362 cm(-1) all undergo shifts of -1 to -2 cm(-1), thereby identifying them as combinations of nu(Fe-S(t)) and delta(Cys). These same four modes undergo similar isotope shifts when T4MOC ferredoxin is selectively labeled with (15)N-histidine ((15)N in either the alpha1,delta1 or delta1,epsilon2 positions). Thus, the Fe-S(Cys) stretch must also be undergoing kinematic coupling with vibrations of the Fe-His moiety. The extensive kinematic coupling of iron ligand vibrations observed in both the Fe(2)S(2) and Rieske ferredoxins presumably arises from the rigidity of the protein framework and is reminiscent of the behavior of cupredoxins. In both cases, the structural rigidity is likely to play a role in minimizing the reorganization energy for electron transfer.