Investigation of the solution conformation of cytochrome c-551 from Pseudomonas stutzeri.

1H NMR spectroscopy and solution structure computations have been used to examine ferrocytochrome c-551 from Pseudomonas stutzeri (ATCC 17588). Resonance assignments are proposed for all main-chain and most side-chain protons. Distance constraints were determined on the basis of nuclear Overhauser enhancements between pairs of protons. Dihedral angle constraints were determined from estimates of scaler coupling constants. Twenty-four structures were calculated by distance geometry and refined by energy minimization and simulated annealing on the basis of 1033 interproton distance and 57 torsion angle constraints. Both the main-chain and side-chain atoms are well defined except for a loop region around residues 34-40, the first two residues at the N-terminus and the last two at the C-terminus, and some side chains located on the molecular surface. The average root mean squared deviation in position for equivalent atoms between the 24 individual structures and the mean structure obtained by averaging their coordinates is 0.54 +/- 0.08 A for the main-chain atoms and 0.97 +/- 0.09 A for all non-hydrogen atoms of residues 3-80 plus the heme group. These structures were compared to the X-ray crystallographic structure of an analogous protein, cytochrome c-551 from Pseudomonas aeruginosa [Matsuura, Takano, & Dickerson (1982) J. Mol. Biol. 156, 389-409). The main-chain folding patterns are very consistent, but there are some differences. The largest difference is in a surface loop segment from residues 34 to 40.     

Solution conformation of the Met 61 to His 61 mutant of Pseudomonas stutzeri ZoBell ferrocytochrome c-551

The gene encoding for bacterial cytochrome c-551 from Pseudomonas stutzeri substrain ZoBell has been mutated to convert the invariant sixth ligand methionine residue into histidine, creating the site-specific mutant M61H. Proton NMR resonance assignments were made for all main-chain and most-side chain protons in the diamagnetic, reduced form at pH 9.2 and 333 K by two-dimensional NMR techniques. Distance constraints (1074) were determined from nuclear Overhauser enhancements and main-chain torsion-angle constraints (72) from scalar coupling estimates. Solution conformations for the protein were computed by the simulated annealing approach. For 28 computed structures, the root mean squared displacement from the average structure excluding the terminal residues 1, 2, 81, and 82 was 0.52 A (sigma = 0.096) for backbone atoms and 0.90 A (sigma = 0.122) for all heavy atoms. The global folding of the mutant protein is the same as for wild type. The biggest changes are localized in a peptide span over residues 60-65. The most striking behavior of the mutant protein is that at room temperature and neutral pH it exists in a state similar to the molten globular state that has been described for several proteins under mild denaturing conditions, but the mutant converts to a more ordered state at high pH and temperature.     

Primary sequence and solution conformation of ferrocytochrome c-552 from Nitrosomonas europaea.

Cytochrome c-552 from Nitrosomonas europaea is a 9.1-kDa monoheme protein that is a member of the bacterial cytochrome c-551 family. The gene encoding for c-552 has been cloned and sequenced and the primary sequence of the product deduced. Proton resonance assignments were made for all main-chain and most side-chain protons in the diamagnetic, reduced form by two-dimensional NMR techniques. Distance constraints (1056) were determined from nuclear Overhauser enhancements, and torsion angle constraints (88) were determined from scalar coupling estimates. Solution conformations for the protein were computed by the hybrid distance geometry-simulated annealing approach. For 20 computed structures, the root mean squared deviation from the average position of equivalent atoms was 0.84 A (sigma = 0.12) for backbone atoms over all residues. Analysis by residue revealed there were three regions clearly less well defined than the rest of the protein: the first two residues at the N-terminus, the last two at the C-terminus, and a loop region from residues 34 to 40. Omitting these regions from the comparison, the root mean squared deviation was 0.61 A (sigma = 0.13) for backbone atoms, 0.86 A (sigma = 0.12) for all associated heavy atoms, and 0. 43 A (sigma = 0.17) for the heme group. The global folding of the protein is consistent with others in the c-551 family. A deletion at the N-terminus relative to other family members had no impact on the global folding, whereas an insertion at residue 65 did affect the way the polypeptide packs against the methionine-ligated side of the heme. The effects of specific substitutions will be discussed. The structure of c-552 serves to delineate essential features of the c-551 family.     

High-resolution solution structure of reduced French bean plastocyanin and comparison with the crystal structure of poplar plastocyanin

The three-dimensional solution structure of reduced (CuI) plastocyanin from French bean leaves has been determined by distance geometry and restrained molecular dynamics methods using constraints obtained from 1H n.m.r. (nuclear magnetic resonance) spectroscopy. A total of 1244 experimental constraints were used, including 1120 distance constraints, 103 dihedral angle constraints and 21 hydrogen bond constraints. Stereospecific assignments were made for 26 methylene groups and the methyls of 11 valines. Additional constraints on copper co-ordination were included in the restrained dynamics calculations. The structures are well defined with average atomic root-mean-square deviations from the mean of 0.45 A for all backbone heavy atoms and 1.08 A for side-chain heavy atoms. French bean plastocyanin adopts a beta-sandwich structure in solution that is similar to the X-ray structure of reduced poplar plastocyanin; the average atomic root-mean-square difference between 16 n.m.r. structures and the X-ray structure is 0.76 A for all backbone heavy atoms. The conformations of the side-chains that constitute the hydrophobic core of French bean plastocyanin are very well defined. Of 47 conserved residues that populate a single chi 1 angle in solution, 43 have the same rotamer in the X-ray structure. Many surface side-chains adopt highly preferred conformations in solution, although the 3J alpha beta coupling constants often indicate some degree of conformational averaging. Some surface side-chains are disordered in both the solution and crystal structures of plastocyanin. There is a striking correlation between measures of side-chain disorder in solution and side-chain temperature factors in the X-ray structure. Side-chains that form a distinctive acidic surface region, believed to be important in binding other electron transfer proteins, appear to be disordered. Fifty backbone amide protons form hydrogen bonds to carbonyls in more than 60% of the n.m.r. structures; 45 of these amide protons exchange slowly with solvent deuterons. Ten hydrogen bonds are formed between side-chain and backbone atoms, eight of which are correlated with decreased proton exchange. Of the 60 hydrogen bonds formed in French bean plastocyanin, 56 occur in the X-ray structure of the poplar protein; two of the missing hydrogen bonds are absent as a result of mutations. It appears that molecular dynamics refinement of highly constrained n.m.r. structures allows accurate prediction of the pattern of hydrogen bonding.     

Solution conformation of the His-47 to Ala-47 mutant of Pseudomonas stutzeri ZoBell ferrocytochrome c-551

In the cytochrome c-551 family, the heme 17-propionate caboxylate group is always hydrogen bonded to an invariant Trp-56 and conserved residues (His and Arg mainly, Lys occasionally) at position 47. The mutation of His-47 to Ala-47 for Pseudomas stutzeri ZoBell cytochrome c-551 removes this otherwise invariant hydrogen bond. The solution structure of ferrous H47A has been solved based on NMR-derived constraints. Results indicate that the mutant has very similar main chain folding compared to wild-type. However, less efficient packing of residues in the mutant surrounding the heme propionates leads to more solvent exposure for both propionate groups, which may account for decreased stability of the mutant. The mutant has a reduction potential different from wild-type, and furthermore, the pH dependence of this potential is not the same as for wild-type. The structure of the mutant suggests that these changes are related to the loss of the residue-47 propionate hydrogen bond and the loss of charge on the side chain of residue 47.     

15N NMR spectroscopy of Pseudomonas cytochrome c-551

15N-1H correlation spectroscopy with detection at the 1H frequency has been used at natural abundance to detect nitrogen nuclei bonded to protons in the ferrocytochrome c-551 from Pseudomonas aeruginosa (ATCC 19429). Side-chain aromatic nitrogens, main-chain amides, and side-chain amides have been assigned to specific residues by comparison to previous proton assignments. Assignment ambiguities arising from overlap in the proton dimension have been resolved by examining spectra as a function of temperature and pH. Nitrogen chemical shifts are reported at pH 4.6 and 9.4 and three temperatures, 32, 50, and 60 degrees C. Significant differences arise from the observed protein shifts and expected shifts in the random coil polypeptide.     

The three-dimensional structure of guanine-specific ribonuclease F1 in solution determined by NMR spectroscopy and distance geometry.

Two-dimensional 1H-NMR studies have been performed on ribonuclease F1 (RNase F1), which contains 106 amino acid residues. Sequence-specific resonance assignments were accomplished for the backbone protons of 99 amino acid residues and for most of their side-chain protons. The three-dimensional structures were constructed on the basis of 820 interproton-distance restraints derived from NOE, 64 distance restraints for 32 hydrogen bonds and 33 phi torsion-angle restraints. A total of 40 structures were obtained by distance geometry and simulated-annealing calculations. The average root-mean-square deviation (residues 1-106) between the 40 converged structures and the mean structure obtained by averaging their coordinates was 0.116 +/- 0.018 nm for the backbone atoms and 0.182 +/- 0.015 nm for all atoms including the hydrogen atoms. RNase F1 was determined to be an alpha/beta-type protein. A well-defined structure constitutes the core region, which consists of a small N-terminal beta-sheet (beta 1, beta 2) and a central five-stranded beta-sheet (beta 3-beta 7) packed on a long helix. The structure of RNase F1 has been compared with that of RNase T1, which was determined by X-ray crystallography. Both belong to the same family of microbial ribonucleases. The polypeptide backbone fold of RNase F1 is basically identical to that of RNase T1. The conformation-dependent chemical shifts of the C alpha protons are well conserved between RNase F1 and RNase T1. The residues implicated in catalysis are all located on the central beta-sheet in a geometry similar to that of RNase T1.     

NMR comparison of prokaryotic and eukaryotic cytochromes c

1H NMR spectroscopy has been used to examine ferrocytochrome c-551 from Pseudomonas aeruginosa (ATCC 19429) over the pH range 3.5-10.6 and the temperature range 4-60 degrees C. Resonance assignments are proposed for main-chain and side-chain protons. Comparison of results for cytochrome c-551 to recently assigned spectra for horse cytochrome c (Wand et al. (1989) Biochemistry 28, 186-194) and mutants of yeast iso-1 cytochrome (Pielak et al. (1988) Eur. J. Biochem. 177, 167-177) reveals some unique resonances with unusual chemical shifts in all cytochromes that may serve as markers for the heme region. Results for cytochrome c-551 indicate that in the smaller prokaryotic cytochrome, all benzoid side chains are rapidly flipping on the NMR time scale. In contrast, in eukaryotic cytochromes there are some rings flipping slowly on the NMR time scale. The ferrocytochrome c-551 undergoes a transition linked to pH with a pK around 7. The pH behavior of assigned resonances provides evidence that the site of protonation is the inner or buried 17-propionic acid heme substituent (IUPAC-IUB porphyrin nomenclature). Conformational heterogeneity has been observed for segments near the inner heme propionate substituent.     

Proton resonance assignments for Pseudomonas aeruginosa ferrocytochrome c-551.

A comparison between two sets of resonance assignments for ferrocytochrome c-551 from Pseudomonas aeruginosa reveals that major differences can be explained by pH effects. In turn, these reveal side chain protonation events in c-551 that markedly influence spectra. The behavior of resonances in a homologous protein from Pseudomonas stutzeri help to clarify ambiguities in the P. aeruginosa case. A corrected and completed set of proline assignments is presented. Labile side chain protons in residue 47, which hydrogen bonds to the inner heme propionate, appear to be in fast exchange with the solvent.     

Solution structure of Fe(II) cytochrome c551 from Pseudomonas aeruginosa as determined by two-dimensional 1H NMR.

The solution structure of Fe(II) cytochrome c551 from Pseudomonas aeruginosa based on 2D 1H NMR data is reported. Two sets of structure calculations were completed with a combination of simulated annealing and distance geometry calculations: one set of 20 structures included the heme-peptide covalent linkages, and one set of 10 structures excluded them. The main-chain atoms were well constrained within the two structural ensembles (1.30 and 1.35 A average RMSD, respectively) except for two regions spanning residues 30-40 and 60-70. The results were essentially the same when global fold comparisons were made between the ensembles with an average RMSD of 1.33 A. In total, 556 constraints were used, including 479 NOEs, 53 volume constraints, and 24 other distances. This report represents the first solution structure determination of a heme protein by 2D 1H NMR and should provide a basis for the application of these techniques to other proteins containing large prosthetic groups or cofactors.     

Determination of the three-dimensional solution structure of the antihypertensive and antiviral protein BDS-I from the sea anemone Anemonia sulcata: a study using nuclear magnetic resonance and hybrid distance geometry-dynamical simulated annealing

The three-dimensional solution structure of the antihypertensive and antiviral protein BDS-I from the sea anemone Anemonia sulcata has been determined on the basis of 489 interproton and 24 hydrogen-bonding distance restraints supplemented by 23 phi backbone and 21 chi 1 side-chain torsion angle restraints derived from nuclear magnetic resonance (NMR) measurements. A total of 42 structures is calculated by a hybrid metric matrix distance geometry-dynamical simulated annealing approach. Both the backbone and side-chain atom positions are well defined. The average atomic rms difference between the 42 individual SA structures and the mean structure obtained by averaging their coordinates is 0.67 +/- 0.12 A for the backbone atoms and 0.90 +/- 0.17 A for all atoms. The core of the protein is formed by a triple-stranded antiparallel beta-sheet composed of residues 14-16 (strand 1), 30-34 (strand 2), and 37-41 (strand 3) with an additional mini-antiparallel beta-sheet at the N-terminus (residues 6-9). The first and second strands of the triple-stranded antiparallel beta-sheet are connected by a long exposed loop (residues 17-30). A number of side-chain interactions are discussed in light of the structure.     

1H NMR studies of human lysozyme: spectral assignment and comparison with hen lysozyme

Complete main-chain (NH and alpha CH) 1H NMR assignments are reported for the 130 residues of human lysozyme, along with extensive assignments for side-chain protons. Analysis of 2-D NOESY experiments shows that the regions of secondary structure for human lysozyme in solution are essentially identical with those found previously in a similar study of hen lysozyme and are in close accord with the structure of the protein reported previously from X-ray diffraction studies in the crystalline state. Comparison of the chemical shifts, spin-spin coupling constants, and hydrogen exchange behavior are also consistent with closely similar structures for the two proteins in solution. In a number of cases specific differences in the NMR parameters between hen and human lysozymes can be correlated with specific differences observed in the crystal structures.     

Determination of the complete three-dimensional structure of the trypsin inhibitor from squash seeds in aqueous solution by nuclear magnetic resonance and a combination of distance geometry and dynamical simulated annealing

The complete three-dimensional structure of the trypsin inhibitor from seeds of the squash Cucurbita maxima in aqueous solution was determined on the basis of 324 interproton distance constraints, 80 non-nuclear Overhauser effect distances, and 22 hydrogen-bonding constraints, supplemented by 27 phi backbone angle constraints derived from nuclear magnetic resonance measurements. The nuclear magnetic resonance input data were converted to the distance constraints in a semiquantitative manner after a sequence specific assignment of 1H spectra was obtained using two-dimensional nuclear magnetic resonance techniques. Stereospecific assignments were obtained for 17 of the 48 prochiral centers of the squash trypsin inhibitor using the floating chirality assignment introduced at the dynamical simulated annealing stage of the calculations. A total of 34 structures calculated by a hybrid distance geometry-dynamical simulated annealing method exhibit well-defined positions for both backbone and side-chain atoms. The average atomic root-mean-square difference between the individual structures and the minimized mean structure is 0.35(+/- 0.08) A for the backbone atoms and 0.89(+/- 0.17) A for all heavy atoms. The precision of the structure determination is discussed and correlated to the experimental input data.     

Heme crevice disorder after sixth ligand displacement in the cytochrome c-551 family

1H NMR and visible absorption spectroscopy were used to monitor sixth ligand methionine displacement reactions in four members of the ferricytochrome c-551 family from Pseudomonas aeruginosa, Pseudomonas stutzeri, Pseudomonas stutzeri substrain ZoBell, and Nitrosomonas europae. Potassium cyanide displaces the methionine ligand with very modest changes in the visible spectra, but profound changes in the NMR spectra. The initial product formed kinetically, designated complex I, changes with time and/or heating to a more thermodynamically favored product termed complex II. Spectra indicate that both I and II are actually a family of closely related conformational isomers. Low temperature NMR spectra of complex II indicate that some of the isomers are in chemical exchange on the NMR time scale. High pH also displaces the methionine ligand in a manner similar to the well-known alkaline transition of mitochondrial cytochrome c. However, the reaction occurs at higher pH values and over a narrower pH range for the c-551 family, and the transition pH range is different for the different proteins studied. The final alkaline forms also show peak widths and a number of peaks indicative of multiple conformational isomers.     

The influence of stereospecific assignments on the determination of three-dimensional structures of proteins by nuclear magnetic resonance spectroscopy. Application to the sea anemone protein BDS-I

The influence of the stereospecific assignments of beta-methylene protons and the classification of chi 1 torsion angles on the definition of the three-dimensional structures of proteins determined from NMR data is investigated using the sea anemone protein BDS-I (43 residues) as a model system. Two sets of structures are computed. The first set comprises 42 converged structures (denoted STEREO structures) calculated on the basis of the complete list of restraints derived from the NMR data, consisting of 489 interproton and 24 hydrogen bonding distance restraints, supplemented by 23 phi backbone and 21 chi 1 side chain torsion angle restraints. The second set comprises 31 converged structures (denoted NOSTEREO structures) calculated from a reduced data set in which those restraints arising from stereospecific assignments, and the corresponding chi 1 torsion angle restraints, are explicitly omitted. The results show that the inclusion of the stereospecific restraints leads to a significant improvement in the definition of the structure of BDS-I, both with respect to the backbone and the detailed arrangement of the side chains. Average atomic rms differences between the individual structures and the mean structures for the backbone atoms are 0.67 +/- 0.12 A and 0.93 +/- 0.16 A for the STEREO and NOSTEREO structures, respectively; the corresponding values for all atoms are 0.90 +/- 0.17 A and 1.17 +/- 0.17 A, respectively. In addition, while the overall fold remains unchanged, there is a small but significant atomic displacement between the two sets of structures.     

Three-dimensional structure of the neurotoxin ATX Ia from Anemonia sulcata in aqueous solution determined by nuclear magnetic resonance spectroscopy

With the aid of 1H nuclear magnetic resonance (NMR) spectroscopy, the three-dimensional structure in aqueous solution was determined for ATX Ia, which is a 46 residue polypeptide neurotoxin of the sea anemone Anemonia sulcata. The input for the structure calculations consisted of 263 distance constraints from nuclear Overhauser effects (NOE) and 76 vicinal coupling constants. For the structure calculation several new or ammended programs were used in a revised strategy consisting of five successive computational steps. First, the program HABAS was used for a complete search of all backbone and chi 1 conformations that are compatible with the intraresidual and sequential NMR constraints. Second, using the program DISMAN, we extended this approach to pentapeptides by extensive sampling of all conformations that are consistent with the local and medium-range NMR constraints. Both steps resulted in the definition of additional dihedral angle constraints and in stereospecific assignments for a number of beta-methylene groups. In the next two steps DISMAN was used to obtain a group of eight conformers that contain no significant residual violations of the NMR constraints or van der Waals contacts. Finally, these structures were subjected to restrained energy refinement with a modified version of the molecular mechanics module of AMBER, which in addition to the energy force field includes potentials for the NOE distance constraints and the dihedral angle constraints. The average of the pairwise minimal RMS distances between the resulting refined conformers calculated for the well defined molecular core, which contains the backbone atoms of 35 residues and 20 interior side chains, is 1.5 +/- 0.3 A. This core is formed by a four-stranded beta-sheet connected by two well-defined loops, and there is an additional flexible loop consisting of the eleven residues 8-18. The core of the protein is stabilized by three disulfide bridges, which are surrounded by hydrophobic residues and shielded on one side by hydrophilic residues.     

Determination of the three-dimensional solution structure of the C-terminal domain of cellobiohydrolase I from Trichoderma reesei. A study using nuclear magnetic resonance and hybrid distance geometry-dynamical simulated annealing

The solution structure of a synthetic 36-residue polypeptide comprising the C-terminal cellulose binding domain of cellobiohydrolase I (CT-CBH I) from Trichoderma reesei was investigated by nuclear magnetic resonance (NMR) spectroscopy. The 1H NMR spectrum was completely assigned in a sequential manner by two-dimensional NMR techniques. A large number of stereospecific assignments for beta-methylene protons, as well as ranges for the phi, psi, and chi 1 torsion angles, were obtained on the basis of sequential and intraresidue nuclear Overhauser enhancement (NOE) and coupling constant data in combination with a conformational data base search. The structure calculations were carried out in an iterative manner by using the hybrid distance geometry-dynamical simulated annealing method. This involved computing a series of initial structures from a subset of the experimental data in order to resolve ambiguities in the assignments of some NOE cross-peaks arising from chemical shift degeneracy. Additionally, this permitted us to extend the stereospecific assignments to the alpha-methylene protons of glycine using information on phi torsion angles derived from the initial structure calculations. The final experimental data set consisted of 554 interproton distance restraints, 24 restraints for 12 hydrogen bonds, and 33 phi, 24 psi, and 25 chi 1 torsion angle restraints. CT-CBH I has two disulfide bridges whose pairing was previously unknown. Analysis of structures calculated with all three possible combinations of disulfide bonds, as well as without disulfide bonds, indicated that the correct disulfide bridge pairing was 8-25 and 19-35. Forty-one structures were computed with the 8-25 and 19-35 disulfide bridges, and the average atomic rms difference between the individual structures and the mean structure obtained by averaging their coordinates was 0.33 +/- 0.04 A for the backbone atoms and 0.52 +/- 0.06 A for all atoms. The protein has a wedgelike shape with an amphiphilic character, one face being predominantly hydrophilic and the other mainly hydrophobic. The principal element of secondary structure is made up of an irregular triple-stranded antiparallel beta-sheet composed of residues 5-9 (beta 1), 24-28 (beta 2), and 33-36 (beta 3) in which strand beta 3 is hydrogen bonded to the other two strands.(ABSTRACT TRUNCATED AT 400 WORDS)     

Solution structure of murine epidermal growth factor determined by NMR spectroscopy and refined by energy minimization with restraints.

The solution structure of murine epidermal growth factor (mEGF) at pH 3.1 and a temperature of 28 degrees C has been determined from NMR data, using distance geometry calculations and restrained energy minimization. The structure determination is based on 730 conformational constraints derived from NMR data, including 644 NOE-derived upper bound distance constraints, constraints on the ranges of 32 dihedral angles based on measurements of vicinal coupling constants, and 54 upper and lower bound constraints associated with nine hydrogen bonds and the three disulfide bonds. The distance geometry interpretation of the NMR data is based on previously published sequence-specific 1H resonance assignments [Montelione et al. (1988) Biochemistry 27, 2235-2243], supplemented here with individual assignments for some side-chain amide, methylene, and isopropyl methyl protons. The molecular architecture of mEGF is the same as that described previously [Montelione et al. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 5226-5230], but the structure is overall more precisely determined by a more extensive set of NMR constraints. Analysis of proton NMR line widths, amide proton exchange rates, and side-chain 3J(H alpha-H beta) coupling constants provides evidence for internal motion in several regions of the mEGF molecule. Because mEGF is one member of a large family of homologous growth factors and protein domains for which X-ray crystal structures are not yet available, the atomic coordinates resulting from the present structure refinement (which we have deposited in the Brookhaven Protein Data Bank) are important data for understanding the structures of EGF-like proteins and for further detailed comparisons of these structures with mEGF.     

Terminal residues in protein chains: residue preference, conformation, and interaction

The known protein structures have been analyzed to find out if there is any pattern in the type of residues used and their conformation at the two terminal positions of the polypeptide chains. While the N-terminal position is overwhelmingly occupied by Met (followed by Ala and Ser), the preference for the C-terminal is not as distinct, the residues with highest propensities being Lys, Arg, Gln, and Asn. Only one main-chain torsion angle, psi, can be defined for the N-terminal residue, which is found to be in the extended conformation due to a favorable electrostatic interaction between the charged amino group and the carbonyl oxygen atom. The distribution of the angle phi for the C-terminal residue, on the other hand, is not much different from that of the nonterminal residues. There are some differences in the distribution of the side-chain torsion angle chi1 of both the terminal residues from the general distribution. The terminal segments are generally flexible and there is a tendency for the more ordered residues to have lesser solvent exposure. About 40% of the terminal groups form a hydrogen bond with protein atoms--a slight preference is observed for the side-chain atoms (more than half of which belong to charged residues) over the main-chain ones. Although the terminal residues are not included in any regular secondary structure, the adjacent ones have a high preference to occur in the beta conformation. There is a higher chance of a beta-strand rather than an alpha-helix to start within the first 6 positions from the N-terminal end. It is suggested that the extended conformation observed for the N-terminal residue propagates along the chain leading to the formation of beta-strand. In the C-terminal end, on the other hand, as one moves upstream the alpha and beta structures are encountered in proportion similar to the average value for these structures in the database. The cleavage site of the zymogen structures has a conformation that can be retained by the N-terminal residue of the active enzyme.     

Primary structure characterization of a Rhodocyclus tenuis diheme cytochrome c reveals the existence of two different classes of low-potential diheme cytochromes c in purple phototropic bacteria

The complete amino acid sequence of a 26-kDa low redox potential cytochrome c-551 from Rhodocyclus tenuis was determined by a combination of Edman degradation and mass spectrometry. There are 240 residues including two heme binding sites at positions 41, 44, 128, and 132. There is no evidence for gene doubling. The only known homolog of Rc. tenuis cytochrome c-551 is the diheme cytochrome c-552 from Pseudomonas stutzeri which contains 268 residues and heme binding sites at nearly identical positions. There is 44% overall identity between the Rc. tenuis and Ps. stutzeri cytochromes with 10 internal insertions and deletions. The Ps. stutzeri cytochrome is part of a denitrification gene cluster, whereas Rc. tenuis is incapable of denitrification, suggesting different functional roles for the cytochromes. Histidines at positions 45 and 133 are the fifth heme ligands and conserved histidines at positions 29, 209, and 218 and conserved methionines at positions 114 and 139 are potential sixth heme ligands. There is no obvious homology to the low-potential diheme cytochromes characterized from other purple bacterial species such as Rhodobacter sphaeroides. There are therefore at least two classes of low-potential diheme cytochromes c found in phototrophic bacteria. There is no more than 11% helical secondary structure in Rc. tenuis cytochrome c-551 suggesting that there is no relationship to class I or class II c-type cytochromes.