Coordination of the heme iron in the low-potential cytochromes c-553 from Desulfovibrio vulgaris and Desulfovibrio desulfuricans. Different chirality of the axially bound methionine in the oxidized and reduced states

The coordination geometry at the heme iron of the cytochromes c-553 from Desulfovibrio vulgaris and Desulfovibrio desulfuricans was investigated by 1H-nuclear magnetic resonance and circular dichroism spectroscopy. Individual assignments were obtained for heme c and the axial ligands. From studies of nuclear Overhauser enhancements the axial histidine imidazole ring orientation relative to the heme group was found to coincide with other c-type cytochromes. In contrast, a new structure was observed for the axial methionine in the reduced cytochromes c-553. This includes S chirality at the iron-bound sulfur atom, but compared to cytochromes c-551 from Pseudomonads and Rhodopseudomonas gelatinosa and cytochrome c5 from Pseudomonas mendocina, which also contain S-chiral methionine, a different spatial arrangement of the gamma- and beta-methylene groups and the alpha carbon of methionine prevails. For the ferricytochromes c-553 R chirality was found for the iron-bound sulfur. This is the first observation of different methionine chirality in different oxidation states of the same c-type cytochrome.     

A new spatial structure for the axial methionine observed in cytochrome c5 from Pseudomonas mendocina. Correlations with the electronic structure of heme c

Cytochrome c5 from Pseudomonas mendocina has been isolated and the coordination geometry at the heme iron was investigated by 1H nuclear magnetic resonance and circular dichroism spectroscopy. Individual assignments were obtained for heme c and the axial ligands. From studies of nuclear Overhauser enhancements the axial histidine imidazole ring orientation relative to the heme group was found to coincide with that of other c-type cytochromes. In contrast, a new structure was observed for the axial methionine. This includes S chirality at the iron-bound sulfur atom, but compared to cytochromes c-551 from Pseudomonads and Rhodopseudomonas gelatinosa, which also contain S-chiral methionine, the spatial arrangement of the gamma- and beta-methylene groups and the alpha carbon of methionine is markedly different. Analysis of the electron spin density distribution in ferricytochrome c5 in the light of this new coordination geometry provides additional support for the hypothesis that the electronic structure of heme c is primarily governed by the orientation of the sp3 lone-pair orbital of the axial sulfur atom with respect to the heme plane.     

An LHRH agonist inhibits FSH-induced cyclic AMP accumulation and steroidogenesis in porcine granulosa cells in culture

The heme iron coordination in horse cytochrome c and cytochrome c-551 from $\text{Pseudomonas aeruginosa}$ was investigated with ¹H NMR and CD spectroscopy. Truncated driven nuclear Overhauser enhancement (TOE) studies of the reduced proteins showed different chirality for the attachment of the axial methionine in the two species. For the oxidized proteins the different chirality was manifested in different CD properties of the 695 nm absorption band. Since additional NMR data indicated nearly identical coordination of the axial histidine in the two species it is suggested that the previously reported different electronic heme structures in the oxidized proteins are a consequence of the different mode of binding of the axial methionine.     

Evolutionary change of the heme c electronic structure: ferricytochrome c-551 from Pseudomonas aeruginosa and horse heart ferricytochrome c.

Individual assignments of the ¹H n.m.r. lines of heme c in reduced and oxidized cytochrome c-551 from $\text{Pseudomonas aeruginosa}$ were obtained by nuclear Overhauser enhancement and saturation transfer experiments. Comparison with the corresponding data on horse heart cytochrome c showed that the locations of high spin density on the heme c periphery as well as the in-plane principal axes x and y of the electronic g-tensor are rotated by approximately 90° in ferricytochrome c-551 relative to horse ferricytochrome c. High spin density in ferricytochrome c-551 is thus localized on the pyrrole ring III. While this pyrrole ring is well shielded in the interior of mammalian-type cytochromes c, it is more easily accessible in cytochrome c-551. It is suggested that this evolutionary change of the heme c electronic structure would be compatible with the hypothesis that the electron transfer in both species is via solvent exposed peripheral ring carbon atoms.     

Structure and heme environment of ferrocytochrome c553 from 1H NMR studies

Cytochrome c553 is a photosynthetic electron transport protein found in algae and cyanobacteria. We have purified cytochromes c553 from five cyanobacteria and studied the structures of the ferrocytochromes by 1H NMR spectroscopy at 360 and 470 MHz. Using standard NMR techniques and by comparing the amino acid sequences of four cytochromes c553 with their 1H NMR spectra, we have assigned in the spectrum of the Aphanizomenon flos-aquae protein 18 resonances to specific amino acid residues and 12 resonances to specific heme protons. Steady state and truncated driven nuclear Overhauser enhancement experiments indicate that a tyrosine and methionine are located near pyrrole ring IV of the heme and that a phenylalanine ring is near the heme alpha-mesoproton. The general folding of the cytochrome c553 protein backbone appears to resemble that of Pseudomonas aeruginosa cytochrome c551, but the chirality of the cytochrome c553 axial methine sulfur is R, the same as that of horse heart cytochrome c.     

Axial ligation and heme environment in cytochrome c-555 from Prosthecochloris aestuarii. Investigation by absorption and solvent perturbation difference spectroscopy.

The near-IR absorption spectrum indicated that methionine is the sixth axial heme iron ligand in Prosthecochloris aestuarii cytochrome c-555. The heme environment has been investigated by the technique of solvent perturbation difference spectroscopy. The heme octapeptide from cytochrome c plus added imidazole was used as a model compound for the fully exposed chromophore. The heme was found to be minimally exposed to solvent. A comparison was made with cytochrome c, as to the possible causes of the difference in redox potentials betweeen these two cytochromes.     

Amino acid sequences of the two heme c-binding sites of Pseudomonas cytochrome-c peroxidase

The amino acid sequences of the two heme c-containing tryptic peptides of Pseudomonas cytochrome-c peroxidase have been determined. The tryptic peptides were isolated from two cyanogen bromide fragments of the protein. Both heme-binding sites have the Cys-X-Y-Cys-His structure characteristic of c-type cytochromes. The sequences of the two peptides show distinct homology with each other, suggesting the occurrence of gene doubling during evolution of the protein molecule. The function of the heme c moieties in the catalytic cycle of the enzyme is discussed on the basis of their homology with the proximal histidine region of peroxidase (horseradish peroxidase and yeast cytochrome-c peroxidase) and cytochromes (horse cytochrome c and Pseudomonas cytochrome c-551).     

Structural basis for the variation of pH-dependent redox potentials of Pseudomonas cytochromes c-551

The redox potentials of many c-type cytochromes vary with pH over the physiological pH range. We have investigated the pH dependence of redox potential for the four homologous cytochromes c-551 from Pseudomonas aeruginosa, Pseudomonas stutzeri strain 221, Pseudomonas stutzeri strain 224, and Pseudomonas mendocina . The pH dependence is due to an ionizable group that ionizes with pKox in ferricytochrome c-551 but with a higher pK, pKred , in ferrocytochrome c-551. For P. aeruginosa cytochrome c-551 it has been shown that this ionizable group is one of the heme propionic acid substituents [Moore, G. R., Pettigrew , G. W., Pitt , R. C., & Williams, R. J. P. (1980) Biochim. Biophys. Acta 590, 261-271]but the values of pKox and pKred are significantly lower in this protein than in the other three cytochromes. NMR and chemical modification studies show that for the two P. stutzeri cytochromes c-551 and P. mendocina cytochrome c-551, this propionic acid substituent is again important for the pH dependence of the redox potential. However, a histidine occurring at position 47 in their sequences hydrogen bonds to the propionic acid and thereby raises its pK. In P. aeruginosa cytochrome c-551, His-47 is substituted by Arg-47. Hydrogen-bonding schemes involving His-47 and the propionic acid are proposed.     

Resilience of Rhodobacter sphaeroides cytochrome bc1 to heme c1 ligation changes

Typically, c hemes are bound to the protein through two thioether bonds to cysteines and two axial ligands to the heme iron. In high-potential class I c-type cytochromes, these axial ligands are commonly His-Met. A change in this methionine axial ligand is often correlated with a dramatic drop in the heme redox potential and loss of function. Here we describe a bacterial cytochrome c with an unusual tolerance to the alternations in the heme ligation pattern. Substitution of the heme ligating methionine (M185) in cytochrome c1 of the Rhodobacter sphaeroides cytochrome bc1 complex with Lys and Leu lowers the redox midpoint potential but not enough to prevent physiologically competent electron transfer in these fully functional variants. Only when Met-185 is replaced with His is the drop in the redox potential sufficiently large to cause cytochrome bc1 electron transfer chain failure. Functional mutants preserve the structural integrity of the heme crevice: only the nonfunctional His variant allows carbon monoxide to bind to reduced heme, indicating a significant opening of the heme environment. This range of cytochrome c1 ligand mutants exposes both the relative resilience to sixth axial ligand change and the ultimate thermodynamic limits of operation of the cofactor chains in cytochrome bc1.     

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.     

Electron transfer proteins of the purple phototrophic bacterium, Rhodopseudomonas rutila.

The soluble electron transfer protein content of Rhodopseudomonas rutila was found to consist of two basic cytochromes and a (4Fe-4S) ferredoxin. Cytochrome c' was easily identified by its characteristic high spin absorption spectra. The native molecular weight is 29,000 and the subunit is 14,000. Cytochrome c-550 has low spin absorption spectra and a high redox potential (376 mV) typical of cytochromes c2. The molecular weight is about 14,000. The ferredoxin is apparently a dimer (43,000) of approximately 18,000 Da subunits. There are 1.3 to 1.5 iron-sulfur clusters per monomer of 18- to 21-kDa protein. The N-terminal amino acid sequence is like the (7Fe-8S) ferredoxins of Rhodobacter capsulatus and Azotobacter vinelandii. Remarkably, there are only 2 or 3 out of 25 amino acid substitutions. Difference absorption spectra of Rps. rutila membranes indicate that there is not tetraheme reaction center cytochrome c, such as is characteristic of Rps. viridis. However, there are a high potential cytochrome c and a low potential cytochrome b in the membrane, which are suggestive of a cytochrome bc1 complex. Rps. rutila is most similar to Rps. palustris in microbiological properties, yet it does not have the cytochromes c-556, c-554, and c-551 in addition to c2 and c', which are characteristic of Rps. palustris. Furthermore, the Rps. rutila cytochrome c' is dimeric, whereas the same protein from Rps. palustris is the only one known to be monomeric. The cytochrome pattern is more like that of Rhodospirillum rubrum and Rb. capsulatus, which are apparently only able to make cytochromes c2 and c'.     

Soluble cytochromes and ferredoxins from the marine purple phototrophic bacterium, Rhodopseudomonas marina

Four soluble c-type cytochromes, the high redox potential 4-Fe-S ferredoxin known as HiPIP, a large molecular weight 2-Fe-S ferredoxin and a 4-Fe-S 'bacterial' ferredoxin, were isolated from extracts of two strains of Rps. marina. Cytochrome c-550, cytochrome c' and cytochrome c-549 were previously described, and we have extended their characterization. Cytochrome c-558, which has not previously been observed in Rps. marina, appears to be a low-spin isozyme of the more commonly observed high-spin cytochrome c'. HiPIP, which was not observed in previous work, was found to be abundant in Rps. marina. The 2-Fe-S ferredoxin, which has previously been observed only in Rps. palustris, has a native size greater than 100 kDa and a subunit size of 17 kDa. The 'bacterial' ferredoxin appears to have only a single four-iron-sulfur cluster. We examined photosynthetic membranes by difference spectroscopy and found abundant c-type cytochromes. Approximately one-quarter of the heme can be reduced by ascorbate and the remainder by dithionite. There is 2 nm difference between the high-potential heme (554 nm) and the low (552 nm). These characteristics resemble those of the tetraheme reaction center cytochrome of Rps. viridis. In addition to the electron transfer components, we found small amounts of a fluorescent yellow protein which has spectral resemblance to a photoactive yellow protein from Ec. halophila.     

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.     

1H NMR studies of the heme iron coordination in cytochrome c-552 from Euglena gracilis.

The coordination of the heme iron in cytochrome c-552 from Euglena gracilis was investigated by 1H NMR studies at 360 MHz. The data imply that the axial heme ligands are His-14 and Met-56 in both the oxidized and the reduced protein. Studies of mixed solutions of ferro- and ferricytochrome c-552, which provided much of the information on the heme structure, also showed that the intermolecular electron exchange is characterized by a bimolecular rate constant of 5-10(6) mol-1-s-1 at 29 degrees C, which is three orders of magnitude faster than the corresponding reaction in solutions of mammalian cytochromes c.     

Amino acid sequence of cytochrome c-552 from a thermophilic hydrogen-oxidizing bacterium, Hydrogenobacter thermophilus.

The complete amino acid sequence of cytochrome c-552 from an extremely thermophilic hydrogen bacterium, Hydrogenobacter thermophilus TK-6 (IAM 12695), was determined. It is a single polypeptide chain of 80 residues, and its molecular weight, including heme, was calculated to be 7,599. The sequence of cytochrome c-552 from H. thermophilus TK-6 closely resembles that of cytochromes c-551 from Pseudomonas species. Moreover, the tertiary structure of Hydrogenobacter cytochrome c-552 is suggested to be similar to that of cytochrome c-551 from Pseudomonas aeruginosa. The sequence similarity between Hydrogenobacter cytochrome c-552 and that of other bacteria physiologically related to H. thermophilus is not high.     

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.     

Sampling field heterogeneity at the heme of c-type cytochromes by spectral hole burning spectroscopy and electrostatic calculations

We report on a comparative investigation of the heme pocket fields of two Zn-substituted c-type cytochromes-namely yeast and horse heart cytochromes c-using a combination of hole burning Stark spectroscopy and electrostatic calculations. The spectral hole burning experiments are consistent with different pocket fields experienced at the hemes of the respective cytochromes. In the case of horse heart Zn-cytochrome c, two distinguishable electronic origins with different electrostatic properties are observed. The yeast species, on the other hand, displays a single electronic origin. Electrostatic calculations and graphics modeling using the linearized finite-difference Poisson-Boltzmann equation performed at selected time intervals on nanosecond-molecular dynamics trajectories show that the hemes of the respective cytochromes sample different potentials as they explore conformational space. The electrostatic potentials generated by the protein matrix at the heme show different patterns in both cytochromes, and we suggest that the cytochromes differ by the number of "electrostatic substates" that they can sample, thus accounting for the different spectral populations observed in the two cytochromes.     

Cytochrome c-550 mediates electron transfer from inducible periplasmic c-type cytochromes to the cytoplasmic membrane of Paracoccus denitrificans

Electron transfer from periplasmic cytochromes c to the membrane-bound respiratory chain has been studied with the isolated cytochromes and membrane preparations from Paracoccus denitrificans. When reduced cytochromes were incubated with spheroplasts only the constitutive cytochrome c-550 was rapidly oxidized. The inducible cytochromes c-551i and c-553i were not oxidized at appreciable rates. Cytochrome c-550 was able to mediate the transfer of electrons from either cytochrome c-551i or cytochrome c-553i to the membrane preparation.     

A comparative study of the resonance Raman spectra of bacterial cytochromes

Resonance Raman spectra were obtained for two newly isolated bacterial cytochromes, Alcaligenes faecalis (ATCC 8750) c554 and Alcaligenes faecalis c556. Their spectra were compared with those of mammalian cytochrome c and two other bacterial cytochromes, Paracoccus denitrificans c550 and Pseudomonas aeruginosa c551. The positions of the Raman bands indicated that, while Al. c554 and Al. c556 were c-type cytochromes with two thioether linkages, several common features found in their Raman spectra were anomalous. These features suggest that the electronic charge density of the porphyrin macrocycle of Al. c554 and Al. c556 is more asymmetric than that of other bacterial and mammalian c-type cytochromes. The Raman evidence indicates that the electronic properties of the heme are controlled by the protein in these two Alcaligenes cytochromes.     

Identification of heme axial ligands of cytochrome c' from Alcaligenes sp. N.C.I.B. 11015

The spectral properties of both ferric and ferrous cytochromes c' from Alcaligenes sp. N.C.I.B. 11015 are reported. The EPR spectra at 77 K and the electronic, resonance Raman, CD and MCD spectra at room temperature have been compared with those of the other cytochromes c' and various hemoproteins. In the ferrous form, all the spectral results at physiological pH strongly indicated that the heme iron(II) is in a high-spin state. In the ferric form, the EPR and electronic absorption spectra were markedly dependent upon pH. EPR and electronic spectral results suggested that the ground state of heme iron(III) at physiological pH consists of a quantum mechanical admixture of an intermediate-spin and a high-spin state. Under highly alkaline conditions, identification of the axial ligands of heme iron(III) was attempted by crystal field analysis of the low-spin EPR g values. Upon the addition of sodium dodecyl sulfate to ferric and ferrous cytochrome c', the low-spin type spectra were induced. The heme environment of this low-spin species is also discussed.