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.

Iron-coproporphyrin III is a natural cofactor in bacterioferritin from the anaerobic bacterium Desulfovibrio desulfuricans.

A bacterioferritin was recently isolated from the anaerobic sulphate-reducing bacterium Desulfivibrio desulfuricans ATCC 27774 [Romão et al. (2000) Biochemistry 39, 6841-6849]. Although its properties are in general similar to those of the other bacterioferritins, it contains a haem quite distinct from the haem B, found in bacterioferritins from aerobic organisms. Using visible and NMR spectroscopies, as well as mass spectrometry analysis, the haem is now unambiguously identified as iron-coproporphyrin III, the first example of such a prosthetic group in a biological system. This unexpected finding is discussed in the framework of haem biosynthetic pathways in anaerobes and particularly in sulphate-reducing bacteria.

Converting a c-type to a b-type cytochrome: Met61 to His61 mutant of Pseudomonas cytochrome c-551.

The gene nirM, coding for cytochrome c-551 in Pseudomonas stutzeri substrain ZoBell, was engineered to mutate Met61, the sixth ligand to the heme c, into His61, thereby converting the typical Met-His coordination of a c-type cytochrome into His-His, typical of b-type cytochromes. The mutant protein was expressed heterologously in Escherichia coli at levels 3-fold higher than in Pseudomonas and purified to homogeneity. The mutant retained low-spin visible spectral characteristics, indicating that the strong field ligand His 61 was coordinated to the iron. The physiochemical properties of the mutant were measured and compared to the wild-type properties. These included visible spectra, ligand binding reactions, stability to temperature and chemical denaturant, oxidation-reduction potentials, and electron-transfer kinetics to the physiological nitrite reductase of Pseudomonas. Despite a change in potential from the normal 260 mV to 55 mV, the mutant retained many of the properties of the c-551 family.

A complex of iron and nucleic acid catabolites is a signal that triggers differentiation in a freshwater protozoan.

The polymorphic ciliated protozoan Tetrahymena vorax can undergo differentiation from the microstomal form, which normally feeds on bacteria and other particulate matter, into the macrostomal cell type, which is capable of ingesting prey ciliates. The process is triggered by exposure of the microstome to an inducer contained in stomatin, an exudate of the prey. To establish the identity of the signal, stomatin was fractionated by combinations of cation exchange, HPLC, and TLC, and the fractions were assayed for biological activity. Although no single active fraction of purified inducer was obtained, all fractions with activity contained ferrous iron and the nucleic acid catabolites hypoxanthine (6-oxypurine) and uracil (2, 4-dioxopyrimidine), probably in a chelated form. The activity of synthetic complexes containing these three components is equivalent to stomatin. These results indicate a role for ferrous iron and its potential in chelated form to signal differentiation in certain protozoa and, perhaps, in other organisms as well.

Solution conformation of ferricytochrome c-551 from Pseudomonas stutzeri substrain ZoBell.

The main chain protons and the majority of side chain protons have been assigned for the ferric form of Pseudomonas stutzeri substrain ZoBell (American Type Culture Collection 14405) cytochrome c-551. The chemical shifts were compared to those for the ferrous protein to determine the pseudocontact shift contribution. These observed values were compared to contributions calculated from the atomic coordinates of the ferrous cytochrome and an optimized effective room temperature g-tensor centered on the paramagnetic ferric iron. The agreement between observed and calculated values indicates that the conformations of the two forms are highly similar.

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.

Resolution of the nirD locus for heme d1 synthesis of cytochrome cd1 (respiratory nitrite reductase) from Pseudomonas stutzeri.

The genetic organization of the nirD locus of Pseudomonas stutzeri ZoBell, necessary for a catalytically active cytochrome cd1 (EC 1.9.3.2), was determined. The locus comprises the unidirectionally transcribed open reading frames nirFDLGH, downstream of nirMC of the nir gene cluster, and immediately upstream of the norCB operon encoding nitric oxide (NO) reductase (EC 1.7.99.7). Notable sequence relatedness was found between NirF and cytochrome cd1 (NirS), within NirDLGH, and between NirM and NirC, suggesting several gene duplication events in this region. The derived NirF protein (391 amino acids, M(r) 43,137) has 23.8% identity (51.1% overall similarity) with NirS, but lacks the N-terminal heme-c-binding domain of NirS. Insertional mutagenesis of the five open reading frames resulted in the loss of respiratory nitrite reductase activity in vivo and in vitro. Mutant strains, when induced with nitrate for denitrification, synthesized a periplasmic cytochrome cd1 lacking heme d1. The defect was caused by the inability of the cell to synthesize heme d1. The nirD locus is proposed to encode a multimeric and multifunctional enzyme complex involved in the synthesis of heme d1. Mutations in nirFDLGH lowered substantially the expression level of norCB. Nir- mutants, unable to generate NO in vivo, provide indirect evidence for an NO sensor and an inducer role of NO for its cognate reductase.

Siroamide: a prosthetic group isolated from sulfite reductases in the genus Desulfovibrio.

While isolating siroheme from enzymes or whole cells of Desulfovibrio species, it was discovered that the main product after metal removal and esterification was not the octamethyl ester derivative of sirohydrochlorin, but a monoamide, heptamethyl ester derivative. The structure of this derivative was established by mass spectrometry and NMR. Nuclear Overhauser enhancement measurements in combination with chemical shift analogy arguments indicate that the 2(1)-acetate has been stereospecifically amidated. Other cellular sources of siroheme were investigated, but only the octamethyl ester derivative was found, with no traces of the amide derivative. The results suggest that, in Desulfovibrio, the physiologically active prosthetic group may be an amidated form of siroheme.

Characteristics of the paramagnetic 1H-NMR spectra of the ferricytochrome c-551 family.

Heme proton resonances have been assigned for ferricytochromes c-551 isolated from four distinct species of bacteria. While the available structure information indicates that the four cytochromes have very similar conformations in solution, including the chirality of the methionine ligand sulfur bond, the chemical shifts of the paramagnetically shifted resonances are surprisingly different, more so than has been previously reported for a homologous series of ferricytochromes. The resonances are contrasted in terms of chemical shift and the temperature dependence of the shift, which gives rise to a very strong anti-Curie effect for some specific protons. Non-methyl heme resonances do display an approximately conserved set of chemical shifts, but the heme methyl groups demonstrate a wide range of values. The 12(1) heme methyl group is always the highest frequency heme methyl, but the relative positions of the other methyl groups may change. The 7(1) heme methyl group always displayed strong anti-Curie behavior, while the 12(1) methyl group displayed normal Curie behavior. The behavior of the other methyl groups was variable. Possible reasons for the range of observations will be discussed. In spite of their NMR differences, all the ferricytochromes c-551 demonstrated comparable electron-transfer rates to a membrane-bound cytochrome reductase system.

Solution conformation of cytochrome c-551 from Pseudomonas stutzeri ZoBell determined by NMR.

1H NMR spectroscopy and solution structure computations have been used to examine ferrocytochrome c-551 from Pseudomonas stutzeri ZoBell (ATCC 14405). Resonance assignments are proposed for all main-chain and most side-chain protons. Stereospecific assignments were also made for some of the beta-methylene protons and valine methyl protons. Distance constraints were determined based upon nuclear Overhauser enhancements between pairs of protons. Dihedral angle constraints were determined from estimates of scalar coupling constants and intra-residue NOEs. Twenty structures were calculated by distance geometry and refined by energy minimization and simulated annealing on the basis of 1012 interproton distance and 74 torsion angle constraints. Both the main-chain and side-chain atoms are well defined except for two terminal residues, and some side-chain atoms located on the molecular surface. The average root mean squared deviation in the position for equivalent atoms between the 20 individual structures and the mean structure obtained by averaging their coordinates is 0.56 +/- 0.10 A for the main-chain atoms, and 0.95 +/- 0.09 A for all nonhydrogen atoms of residue 3 to 80 plus the heme group. The average structure was compared with an analogous protein, cytochrome c-551 from pseudomonas stutzeri. The main-chain folding patterns are very consistent, but there are some differences, some of which can be attributed to the loss of normally conserved aromatic residues in the ZoBell c-551.

Investigation of the structure of oxidized Pseudomonas aeruginosa cytochrome c-551 by NMR: comparison of observed paramagnetic shifts and calculated pseudocontact shifts.

Extensive main-chain and side-chain assignments are reported for the 1H NMR spectrum of ferricytochrome c-551 from Pseudomonas aeruginosa at 323 K and pH 5.2. These were obtained by sequential assignments of two-dimensional scalar and dipolar correlation spectra. The low-spin (S = 1/2) ferric iron in the oxidized state gives rise to extensive contact and pseudocontact shifts for resonances in the ferricytochrome. Total redox-state-dependent shifts were computed by comparison to the previously assigned ferrocytochrome c-551. A set of 179 firmly assigned protons was selected that were expected to experience only pseudocontact shift contributions in the oxidized form. The pseudocontact shifts were calculated for the set by a standard model [Williams, G., Clayden, N. J., Moore, G. R., & Williams, R. J. P. (1985) J. Mol. Biol. 183, 447-460] using the atomic coordinates from the X-ray crystallographic determination of the oxidized form [Matsuura, Y., Takano, T., & Dickerson, R. E. (1982) J. Mol. Biol. 156, 389-409], and effective anisotropy and geometric factors were adjusted to minimize the sum of the squared differences between observed redox-state shifts and calculated pseudocontact shift contributions. The root-mean-squared deviation with the optimized parameters was 0.12 ppm. The optimized model was then used to calculate pseudocontact shifts for other assigned protons outside the basic set. The overall agreement and the lack of any systematic discrepancies provide evidence that there are no major structural differences among the solution and crystal conformations of the oxidized and reduced forms, within the inherent resolution of this computational approach.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of heme c peptides by mass spectrometry.

Liquid secondary ion mass spectrometry, also called fast atom bombardment mass spectrometry, has been used to determine proteolytic hydrolysis sites for peptides derived from c-type cytochromes that contain covalently attached heme c. An unexpected fragmentation occurs that breaks the covalent thioether bonds between the heme and the peptide, and the heme fragment further rearranges to give rise to an intense ion at m/z of 617 that corresponds to protonated iron protoporphyrin IX. The observation of this ion fragment can be used to unambiguously identify the presence of authentic heme c in peptides or peptide mixtures at the subnanomole level.

Evidence for the structure of the active site heme P460 in hydroxylamine oxidoreductase of Nitrosomonas.

Hydroxylamine oxidoreductase (HAO) is responsible for the oxidation of hydroxylamine to nitrite in nitrification by Nitrosomonas europaea. It has an alpha n subunit structure and eight covalently bound hemes per subunit. Seven of these have visible spectra indistinguishable from heme c. The eighth, designated as P460, has unusual visible spectroscopic features in the enzyme and in a heme-containing proteolytic fragment. Its structure has not been previously determined. Enzymatic digestions of HAO were performed, and various proteolytic fragments were purified. Mass spectrometry confirmed the presence of authentic heme c in some fragments, that is, iron protoporphyrin IX cross-linked by two thioether bonds to cysteine residues. It was possible to detect the presence of the P460 pigment in some fragments, based upon the sensitivity of this pigment to treatment of the holoenzyme with hydrogen peroxide. A proteolytic fragment produced by sequential digestion with trypsin and pronase was shown to contain heme c and a hydrogen peroxide-sensitive heme with an unusual visible spectrum. This fragment contained two covalently cross-linked peptides. Mass spectrometry and NMR indicated that the P460 heme was iron protoporphyrin IX covalently bonded by two thioether bridges to peptide, but in addition there was a new, third covalent bond between a meso heme carbon and an aromatic ring carbon on a tyrosyl residue. The new covalent bond has been tentatively assigned to the C2 carbon of the tyrosyl ring and the 5-meso heme carbon (IUPAC-IUB tetrapyrrole nomenclature), although this location requires further proof.

Ionization of the heme propionate substituents in pseudomonad cytochromes c-551.

The heme propionate substituents in Pseudomonas cytochrome c-551 are partially buried by folds of polypeptide in the structure of the protein, and are involved in several hydrogen bonds. The ionization behavior of these groups has been of interest because the oxidation potential of the heme changes with pH in a manner that may parallel ionization of a propionate. The ionization pKa's of these groups have been determined by following the NMR chemical shifts of nearby protons acting as probes of the ionization state of the propionates. In Pseudomonas aeruginosa c-551 the 13-propionate (IUB-IUPAC porphyrin nomenclature) has been assigned a pKa of 3.1, and the 17-propionate a pKa of 7.2. In the homologous Pseudomonas stutzeri c-551, the respective propionates both have pKa values of 3.0.

Identification of heme macrocycle type by near-infrared magnetic circular dichroism spectroscopy at cryogenic temperatures.

The electron paramagnetic resonance (EPR) and near-infrared magnetic circular dichroism (MCD) spectra of the azide and cyanide adducts of nitrimyoglobin and hydroperoxidase II from Escherichia coli have been measured at cryogenic temperatures. For the first time, ligand-to-metal charge-transfer transitions in the near-infrared have been observed for an Fe(III)-chlorine system. It is shown that near-ultraviolet-to-visible region electronic spectra of 'green' hemes such as these are an unreliable indicator of macrocycle type. However, the combined application of EPR and near-infrared MCD spectroscopies clearly distinguishes between the porphyrin-containing nitrimyoglobin and the chlorine-containing hydroperoxidase II.

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.

Hydrogen exchange in Pseudomonas cytochrome c-551.

Hydrogen exchange rates were measured or estimated for 75 amide protons in in ferrocytochrome c-551 from Pseudomonas aeruginosa (82 residues total) at neutral pH and 300 K. Rate constants span at least eight orders of magnitude. Rate constants or limiting estimates were determined by a combination of methods relying upon 1H-NMR spectroscopy, including the direct observation in one- or two-dimensional spectra of the decrease in proton intensity for samples dissolved in deuterium oxide, or, in a few favorable cases, saturation transfer from the solvent protic water. The heme ligand residues and the thioether bridge residues were slowly exchanging backbone amides, but the slowest exchanging backbone amides were found in two clusters. One was composed of Ile-48 and Lys-49 in the last turn of what is termed the 40's helix in the protein. The second was composed of Leu-74, Ala-75, Lys-76 and Val-78 in the C-terminal alpha helix.

A novel derivative of the prosthetic group heme d1: S-methylporphyrindione d1.

Several derivatives of heme d1 have been characterized by ultraviolet-visible, NMR, and mass spectrometry. Most arise from side reactions during the isolation of d1 from the enzyme. One, however, has now been shown to correspond to the replacement of a meso proton by an S-methyl group. Since the porphyrin is not exposed to S-methyl-containing reagents during its isolation, this raises hypotheses that it has its origin in vivo.

Hierarchy of globin complexes. The quaternary structure of the extracellular chlorocruorin of Eudistylia vancouverii.

The molecular dimensions of the extracellular, hexagonal bilayer chlorocruorin of the polychaete Eudistylia vancouverii, determined by scanning transmission electron microscopy (STEM) of negatively stained specimens, were diameter of 27.5 nm and height of 18.5 nm. STEM mass measurements of unstained, freeze-dried specimens provided a molecular mass (Mm) of 3480 +/- 225 kDa. The chlorocruorin had no carbohydrate and its iron content was 0.251 +/- 0.021 wt%, corresponding to a minimum Mm of 22.4 kDa. Mass spectra and nuclear magnetic resonance spectra of the prosthetic group confirmed it to be protoheme IX with a formyl group at position 3. SDS/polyacrylamide gel electrophoresis, reversed-phase chromatography and N-terminal sequencing suggested that the chlorocruorin consists of at least three chains of approximately 30 kDa and five chains of approximately 16 kDa; the two types of subunits occur in the ratio 0.26:0.74(+/- 0.08). Complete dissociation of the chlorocruorin at neutral pH in the presence of urea or guanidine hydrochloride, followed by gel filtration, produced elution profiles consisting of three peaks, B, C and D. Fractions B and C consisted of the approximately 16 kDa chains and fraction D consisted of the approximately 30 kDa subunits. Mass measurements of particles in STEM images of unstained, freeze-dried fractions B and C provided Mm of 208 +/- 23 kDa and 65 +/- 12 kDa, respectively, in agreement with 191 +/- 13 kDa and 67 +/- 5 kDa obtained by gel filtration. Particles with Mm = 221 +/- 21 kDa were also observed in STEM images of unstained, freeze-dried chlorocruorin. These results imply that the chlorocruorin structure, in addition to the approximately 30 kDa linker subunits that have 0.26 to 0.47 heme groups/chain, comprises approximately 65 kDa tetramers and approximately 200 kDa dodecamers (trimers of tetramers) of globin chains. The stoichiometry of the tetramer and linker subunits calculated from molar amino acid compositions was 34 +/- 4 and 43 +/- 9. The complete dissociation of the chlorocruorin was accompanied by a 50 to 75% loss of the 55 +/- 14 Ca2+/mol protein, and was decreased to approximately 35% by the presence of 10 to 25 mM-Ca2+. Reassociation of dissociated chlorocruorin was maximal in the presence of 2.5 to 5 mM-Ca2+. The dodecamer and/or tetramer subunits in the absence or presence of Ca2+ exhibited very limited (less than 10%) reassociation into hexagonal bilayer structures, only in the presence of the linker subunit.(ABSTRACT TRUNCATED AT 400 WORDS)

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.