Homogeneous Cytochrome 579 Is an Octamer That Reacts Too Slowly With Soluble Iron to Be the Initial Iron Oxidase in the Respiratory Chain of .

The exact role that cytochrome 579 plays in the aerobic iron respiratory chain of is unclear. This paper presents genomic, structural, and kinetic data on the cytochrome 579 purified from cell-free extracts of cultured on soluble iron. Electrospray mass spectrometry of electrophoretically homogeneous cytochrome 579 yielded two principal peaks at 16,015 and 16,141 Daltons.

Solution structure of the Cys74 to Ala74 mutant of the recombinant catalytic domain of Zoocin A.

Zoocin A is a Zn-metallopeptidase secreted by Streptococcus zooepidemicus strain 4881. Its catalytic domain is responsible for cleaving the D-alanyl-L-alanine peptide bond in streptococcal peptidoglycan. The solution NMR structure of the Cys74 to Ala74 mutant of the recombinant catalytic domain (rCAT C74A) has been determined.

Proposed docking interface between peptidoglycan and the target recognition domain of zoocin A.

A docking model is proposed for the target recognition domain of the lytic exoenzyme zoocin A with the peptidoglycan on the outer cell surface of sensitive bacterial strains. Solubilized fragments from such peptidoglycans perturb specific backbone and side chain amide resonances in the recombinant form of the domain designated rTRD as detected in two-dimensional (1)H-(15)N correlation NMR spectra. The affected residues comprise a shallow surface cleft on the protein surface near W115, N53, N117, and Q105 among others, which interacts with the peptide portion of the peptidoglycan.

His86 from the N-terminus of frataxin coordinates iron and is required for Fe-S cluster synthesis.

Human frataxin has a vital role in the biosynthesis of iron-sulfur (Fe-S) clusters in mitochondria, and its deficiency causes the neurodegenerative disease Friedreich's ataxia. Proposed functions for frataxin in the Fe-S pathway include iron donation to the Fe-S cluster machinery and regulation of cysteine desulfurase activity to control the rate of Fe-S production, although further molecular detail is required to distinguish these two possibilities. It is well established that frataxin can coordinate iron using glutamate and aspartate side chains on the protein surface; however, in this work we identify a new iron coordinating residue in the N-terminus of human frataxin using complementary spectroscopic and structural approaches.

Solution structure of the recombinant target recognition domain of zoocin A.

The protein rTRD is the recombinant form of the target recognition domain of zoocin A, a lytic exoenzyme produced by Streptococcus equi subspecies zooepidemicus 4881. It has no known sequence homologs. However, the catalytic domain of zoocin A is homologous to lysostaphin which is another exoenzyme active against a different spectrum of bacteria, including the pathogen Staphylococcus aureus.

Use of 4-sulfophenyl isothiocyanate labeling and mass spectrometry to determine the site of action of the streptococcolytic peptidoglycan hydrolase zoocin A.

Zoocin A is a streptococcolytic peptidoglycan hydrolase with an unknown site of action that is produced by Streptococcus equi subsp. zooepidemicus 4881. Zoocin A has now been determined to be a d-alanyl-l-alanine endopeptidase by digesting susceptible peptidoglycan with a combination of mutanolysin and zoocin A, separating the resulting muropeptides by reverse-phase high-pressure liquid chromatography, and analyzing them by mass spectrometry (MS) in both the positive- and negative-ion modes to determine their compositions.

The metal binding site of zoocin A.

Direct metal analysis of the bacteriolytic exoenzyme zoocin A failed to unequivocally identify a putative metal cofactor; hence, indirect experiments utilizing NMR were undertaken to settle this question. Cd(2+) as a surrogate metal ion was reconstituted into EDTA-treated, metal-free recombinant zoocin, and (113)Cd-NMR was employed to explore binding in the protein for this ion. The Cd-substituted enzyme was found to have 80-85% of native streptococcolytic activity.

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.

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.

Siro(haem)amide in Allochromatium vinosum and relevance of DsrL and DsrN, a homolog of cobyrinic acid a,c-diamide synthase, for sulphur oxidation.

In the purple sulphur bacterium Allochromatium vinosum, the prosthetic group of dissimilatory sulphite reductase (DsrAB) was identified as siroamide, an amidated form of the classical sirohaem. The genes dsrAB are the first two of a large cluster of genes necessary for the oxidation of sulphur globules stored intracellularly during growth on sulphide and thiosulphate. DsrN is homologous to cobyrinic acid a,c diamide synthase and may therefore catalyze glutamine-dependent amidation of sirohaem.

Mutation of the putative hydrogen-bond donor to P700 of photosystem I.

The primary electron donor of photosystem I (PS1), called P(700), is a heterodimer of chlorophyll (Chl) a and a'. The crystal structure of photosystem I reveals that the chlorophyll a' (P(A)) could be hydrogen-bonded to the protein via a threonine residue, while the chlorophyll a (P(B)) does not have such a hydrogen bond. To investigate the influence of this hydrogen bond on P(700), PsaA-Thr739 was converted to alanine to remove the H-bond to the 13(1)-keto group of the chlorophyll a' in Chlamydomonas reinhardtii.

Compound 800, a natural product isolated from genetically engineered Pseudomonas: proposed structure, reactivity, and putative relation to heme d1.

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.

NMR evidence for independent domain structures in zoocin A, an antibacterial exoenzyme.

NMR was used to obtain spectroscopic evidence supporting a two domain model for zoocin A in which an N-terminal catalytic domain is linked by a threonine-proline rich linker to a target recognition domain responsible for recognizing the cell wall of bacteria susceptible to the bacteriolytic action of the enzyme. When cloned and separately expressed, each domain retains the folding found in the whole enzyme. Additionally, spectroscopy suggests that the target recognition domain has a conformation typical of a soluble globular protein, while the catalytic domain aggregates at low millimolar concentrations.

Expression of Pseudomonas stutzeri Zobell cytochrome c-551 and its H47A variant in Escherichia coli.

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.

Identification of hemes and related cyclic tetrapyrroles by matrix-assisted laser desorption/ionization and liquid secondary ion mass spectrometry.

Mass spectrometry has proven to be a powerful technique applicable on trace amounts for the identification of known hemes and cyclic tetrapyrroles, and for providing critical information for the structure of new and novel versions. This report describes investigations of the practical limits of detection for such bioinorganic prosthetic groups, primarily by liquid secondary ion mass spectrometry (LSIMS) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS), including a survey of the utility of common matrices. The lower limit of detection under favorable conditions extends to low picomole amounts.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.