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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.

Genetically engineered strains of Escherichia coli and Pseudomonas aeruginosa were prepared harboring the gene cluster nirFDLGH from Pseudomonas stutzeri substrain ZoBell on a high copy plasmid. These genes have been previously implicated as being essential for the biosynthesis of heme d(1), the prosthetic group of dissimilatory nitrite reductases in anaerobic, denitryfying bacteria. Tetrapyrroles detectable at steady-state levels were identified from both organisms, and cell-free extracts from each were also used to transform uroporphyrinogen in vitro. E. coli does not naturally produce d(1), and the engineered strain failed to produce d(1) or any tetrapyrrole foreign to E. coli. Therefore, while nirFDLGHmay be necessary for d(1) biosynthesis, it is not sufficient. In the denitrifier P. aeruginosa, the results were more positive. The presence of the plasmid led to increased levels of d(1). In addition, a previously unidentified tetrapyrrole was detected. This compound was characterized by visible absorption spectroscopy, infrared spectroscopy, X-ray photoelectron spectroscopy, mass spectrometry, and NMR, and a tentative structure was proposed for this compound. The tetrapyrrole has structural features similar to sirohydrochlorin (as precorrin-2 or sirotetrahydrochlorin, a known intermediate of d(1)) and d(1) itself. The most unusual substituents are epoxide and sulfoxide moieties. When this tetrapyrrole was treated with strong mineral acid and heat, it was converted into natural d(1).

The nirM gene encoding cytochrome c-551 from Pseudomonas stutzeri Zobell (PZ) has been expressed in Escherichia coli at levels higher than those previously reported but only under strict anaerobic growth conditions. Expression yields for wild-type cytochrome in this study typically reached 0.6 micromol per liter of saturated E. coli culture (5.5mg/L). Culture conditions investigated are compared to obtained c-551 expression levels; the results may lead to a greater understanding of the challenges encountered when expressing c-type hemoproteins in E. coli. The nirM gene was mutated to produce a histidine-47-alanine mutation of c-551 that been heterologously expressed in E. coli using optimum culture conditions and had its physiochemical properties compared to those of the wild-type protein. In PZ, the histidine-47 residue is part of a conserved hydrogen-bonding network located at the bottom of the heme crevice that also involves tryptophan-56 and a heme propionate. Ionization events within this network are experimentally demonstrated to modulate c-551 oxidation-reduction potential and its observed dependence on pH around neutrality. The redox potential of the mutant cytochrome still displays pH-dependence; however, the midpoint potential is approximately 25mV lower with respect to wild-type c-551 at neutral pH while the pK at which the heme propionate (HP-17) ionizes is lowered by 1.3 pH units. Temperature and chemical denaturant studies also show that loss of the hydrogen-bond-donating imidazole leads to a large decrease in c-551 tertiary stability.

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.

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.

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., (Copyright 1999 Academic Press.)

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.

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.

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.

The dissimilatory nitrite reductase, cytochrome cd1, from Pseudomonas aeruginosa (ATCC 19429) was irreversibly inactivated by methyl- or phenylhydrazine but was only reduced by hydrazine itself. The reaction required oxygen and several turnovers, approximately four, of the cytochrome acting to transfer reducing equivalents from phenylhydrazine to oxygen. The reaction with methyl- or phenylhydrazine altered the visible spectrum of the cytochrome. Bands characteristic of reduced heme c appeared plus new features that were not characteristic of either oxidized or reduced heme d1. Extraction of the heme from phenylhydrazine-treated cytochrome yielded a covalently modified form of the original heme d1. Visible, 1H NMR, and mass spectra were obtained on the purified modified heme and on the metal-free esterified derivative. The spectroscopic data indicate that the modification was the regiospecific substitution of the 5 meso-proton by a phenyl group.

Product inhibition has been examined in the turnover kinetics of cytochrome cd1 from Pseudomonas aeruginosa (ATCC 19429) and from Paracoccus denitrificans1 (ATCC 13456). A common characteristic was a decrease in rate during the time course of assays that was not due to substrate depletion or irreversible inactivation. The product of nitrite reduction, nitric oxide (NO), acted as a product inhibitor in anaerobic assays with an apparent Ki of 0.2 microM, but only if the enzyme was first preincubated with NO for 15 min. The enzyme was inhibited by the oxidized form of electron donors and this could account for the decrease in rate during an assay. For the donors hydroquinone, ascorbate, TMPD, and azurin, measured values of the inhibition constant were at least ten fold lower than measured Km's. Cytochromes c as donors demonstrated a complex pattern of product inhibition by the ferric form. Although numerical values of Ki in these cases were not obtained, trends indicated that apparent values would be less than Km.

Electron self-exchange has been measured by an NMR technique for cytochromes c551 from Pseudomonas aeruginosa and Pseudomonas stutzeri. The rate for P. aeruginosa cyt c551 is 1.2 x 10(7) M-1 s-1 at 40 degrees C in 50 mM phosphate at pH 7. For P. stutzeri, under the same conditions, the rate is 4 x 10(7) M-1 s-1. For both cytochromes, the rate was independent of ionic strength up to 0.5 M in added NaC1, the enthalpy of activation was 20 +/- 4 kcal mol-1, and the entropy of activation was 38 +/- 10 cal mol-1 deg-1.