Graham W. Pettigrew

Haem staining in gels, a useful tool in the study of bacterial c-type cytochromes

Abstract c -Type cytochromes are the only cytochromes to retain quantitatively their haem during SDS gel electrophoresis and can be identified in complex mixtures by their haem peroxidase activity. Although weak staining bands may be due to residual haem attachment to b -type cytochromes or to migration of haem, these effects could be abolished by prior extraction with organic solvent. The colour yield of haem staining allowed an estimate of the relative amounts of a particular cytochrome, particularly if loadings were below 50 pmol. At greater loadings, a plateau of colour development was observed. Freshly made gels gave much poorer colour development. The haem-staining method was shown to be useful in three particular areas of study in bacterial respiration. Firstly, it allows assessment of the results of sphaeroplast formation in gram negative bacteria. Secondly, quantitation of the haem stain was useful in the investigation of the induction effects of growth conditions on c -type cytochromes. Thirdly, the interpretation of complex chromatographic profiles was greatly simplified by the use of haem-stained SDS electrophoretic gels.

pH dependence of the redox potential of Pseudomonas aeruginosa cytochrome c-551

The redox potential of Ps. aeruginosa cytochrome c-551 varies with pH between pH 5 and 8. The pH dependence can be analysed in terms of a pKa of 6.2 in the oxidised form and a pKa of 7.3 in the reduced form. The same pKa values are also observed in NMR spectra of the two oxidation states and the pKa of 7.3 is observed in titration of the visible absorption spectrum of the ferrocytochrome. From the NMR studies these pKa values have been assigned to the ionisation of one of the haem propionic acid groups. pH dependence of redox potential is of variable occurrence among cytochromes and the possible significance and basis of this variation is discussed.

The amino acid sequence of a cytochrome c from a protozoan Crithidia oncopelti

in all of the cytochromes c studied from higher organisms. Some work has been done on the unicellular eukaryotes, protozoa and algae [l-3] , but no sequence has been published. Two kinds of c-type cytochromes are distinguished, those presumed to be members of the above group of mitochondrial proteins and those presumed to be connected with photosynthesis e.g. cytochrome C-552 of Euglena. Both groups may be interesting for the attempts to construct molecular phylogenies and also in the relation of structure with function and properties.

Characterisation of ionisations that influence the redox potential of mitochondrial cytochrome c and photosynthetic bacterial cytochromes c2

Abstract Several cytochromes c 2 from the Rhodospirillaceae show a pH dependence of redox potential in the physiological pH range which can be described by equations involving an ionisation in the oxidised form (p K o ) and one in the reduced form (p K r ). These cytochromes fall into one of two groups according to the degree of separation of p K o and p K r . In group A, represented here by the Rhodomicrobium vannielii cytochrome c 2 , the separation is approx. one pH unit and the ionisation is that of a haem propionic acid. Members of this group are unique among both cytochromes c 2 and mitochondrial cytochromes c in lacking the conserved residue Arg-38. We propose that the role of Arg-38 is to lower the p K of the nearby propionic acid, so that it lies out of the physiological pH range. Substitution of this residue by an uncharged amino acid leads to a raised p K for the propionic acid. In group B, represented here by Rhodopseudomonas viridis cytochrome c 2 , the separation between p K o and p K r is approx. 0.4 pH unit and the ionisable group is a histidine at position 39. This was established by NMR spectroscopy and confirmed by chemical modification. Only a few other members of the cytochrome c 2 /mitochondrial cytochrome c family have a histidine at this position and of these, both Crithidia cytochrome c -557 and yeast cytochrome c were found to have a pH-dependent redox potential similar to that of Rps. viridis cytochrome c 2 . Using Coulombs law, it was found that the energy required to separate p K o and p K r could be accounted for by simple electrostatic interactions between the haem iron and the ionisable group.

Unusual redox behaviour of cytochrome b-561 from bovine chromaffin granule membranes

Redox titrations of cytochrome b-561 have been performed with the purified cytochrome and with intact and detergent-solubilized chromaffin-granule membranes. The midpoint redox potential of the cytochrome is 100-130 mV; this depends upon the composition of the buffer, but is independent of pH in the range 5.5-7.5; partial proteolysis of the cytochrome raises the midpoint potential to 160 mV. The Nernst plots of titration data have slopes of 75-115 mV, and are in some cases sigmoid in shape. This may be explained by negative cooperativity during redox transitions in oligomeric cytochrome b-561. Measurements of the haem and cytochrome content of chromaffin granule membrane suggest a haem content of 1 mol/mol protein. Chemical crosslinking of cytochrome b-561 suggests that it may exist as an oligomer of 4-6 polypeptide chains within the chromaffin granule membrane. Aggregation of purified cytochrome b-561 was shown by gel filtration studies and by immunological methods in SDS-polyacrylamide gels. Studies of the molecular weight of the aggregates suggest that the monomer has a molecular weight close to 22 000, but migrates anomalously slowly during electrophoresis.

A cytochrome c peroxidase from Pseudomonas nautica 617 active at high ionic strength: expression, purification and characterization.

Cytochrome c peroxidase was expressed in cells of Pseudomonas nautica strain 617 grown under microaerophilic conditions. The 36.5 kDa dihaemic enzyme was purified to electrophoretic homogeneity in three chromatographic steps. N-terminal sequence comparison showed that the Ps. nautica enzyme exhibits a high similarity with the corresponding proteins from Paracoccus denitrificans and Pseudomonas aeruginosa. UV-visible spectra confirm calcium activation of the enzyme through spin state transition of the peroxidatic haem. Monohaemic cytochrome c(552) from Ps. nautica was identified as the physiological electron donor, with a half-saturating concentration of 122 microM and allowing a maximal catalytic centre activity of 116,000 min(-1). Using this cytochrome the enzyme retained the same activity even at high ionic strength. There are indications that the interactions between the two redox partners are mainly hydrophobic in nature.

Structural and mutagenesis studies on the cytochrome c peroxidase from Rhodobacter capsulatus provide new insights into structure-function relationships of bacterial di-heme peroxidases.

Cytochrome c peroxidases (CCP) play a key role in cellular detoxification by catalyzing the reduction of hydrogen peroxide to water. The di-heme CCP from Rhodobacter capsulatus is the fastest enzyme (1060 s-1), when tested with its physiological cytochrome c substrate, among all di-heme CCPs characterized to date and has, therefore, been an attractive target to investigate structure-function relationships for this family of enzymes. Here, we combine for the first time structural studies with site-directed mutagenesis and spectroscopic studies of the mutant enzymes to investigate the roles of amino acid residues that have previously been suggested to be important for activity. The crystal structure of R. capsulatus at 2.7 Å in the fully oxidized state confirms the overall molecular scaffold seen in other di-heme CCPs but further reveals that a segment of about 10 amino acids near the peroxide binding site is disordered in all four molecules in the asymmetric unit of the crystal. Structural and sequence comparisons with other structurally characterized CCPs suggest that flexibility in this part of the molecular scaffold is an inherent molecular property of the R. capsulatus CCP and of CCPs in general and that it correlates with the levels of activity seen in CCPs characterized, thus, far. Mutagenesis studies support the spin switch model and the roles that Met-118, Glu-117, and Trp-97 play in this model. Our results help to clarify a number of aspects of the debate on structure-function relationships in this family of bacterial CCPs and set the stage for future studies.

Structural studies by proton NMR of cytochrome c-557 from Crithidia oncopelti

A vast amount of information has been accumulated about the amino acid sequences of cytochromes c from different species [ 11. In addition to their phylogenetic importance, these data are of great interest for the investigation of structure-function relations in cytochrome c. From the primary structures of the proteins of over 30 species it had been inferred that the amino acids involved in the fixation of the heme group, i.e. Cys-14, Cys-17, His-18, and Met-80 [2-71 are among the invariant residues required for the proper cytochrome c function. More recently two cytochromes c from protozoans were found to have Cys-14 replaced by Ala, yet otherwise to be homologous with other eukaryotic cytochromes c [g-lo]. the heme in these proteins is probably covalently linked with the remaining Cys-17, whereas the second vinyl side chain is free [8]. It is of course of much interest to investigate how this apparently important variation affects other properties of the proteins. In this paper the proton NMR features of cytochrome c-557 from Crithidia oncopelti are reported and compared with those of other cytochromes c. Proton NMR spectroscopy has in recent years been found to be a suitable technique for structural studies of cytochromes c [4, 5, 7, 11, 121, e.g. in the identification of methionine as the sixth ligand of the heme iron [4, 5, 71. Outstanding NMR spectral features are conserved in cytochromes c from different species both

The Structure of an Electron Transfer Complex Containing a Cytochrome c and a Peroxidase

Efficient biological electron transfer may require a fluid association of redox partners. Two noncrystallographic methods (a new molecular docking program and 1H NMR spectroscopy) have been used to study the electron transfer complex formed between the cytochrome c peroxidase (CCP) ofParacoccus denitrificans and cytochromes c. For the natural redox partner, cytochrome c 550, the results are consistent with a complex in which the heme of a single cytochrome lies above the exposed electron-transferring heme of the peroxidase. In contrast, two molecules of the nonphysiological but kinetically competent horse cytochrome bind between the two hemes of the peroxidase. These dramatically different patterns are consistent with a redox active surface on the peroxidase that may accommodate more than one cytochrome and allow lateral mobility.

The microaerophilic respiration of Campylobacter mucosalis.

A model is proposed for the respiratory adaptation to falling oxygen concentration during growth of the microaerophilic bacterium Campylobacter mucosalis. During the early stages of growth, the oxidation of formate is a two-stage branched process involving the production of H2O2 followed by its peroxidatic removal. In later stages of growth, at lower oxygen concentrations, the predominant electron flow is linear to a membrane-bound cytochrome-c oxidase which reduces O2 directly to H2O. Several components of this model have been investigated. H2O2 was produced during formate oxidation and accumulated when electron transfer to the cytochrome-c peroxidase was inhibited. A cytochrome c-553, of the Class 1 types, was purified and shown to be the specific electron donor to both the peroxidase and the membrane-bound oxidase. The levels of this cytochrome c and of the peroxidase were higher in cells harvested early in growth. In later stages of growth, the activity of the membrane-bound oxidase increased. Proton pumping across the membrane was detected with either H2O2 or oxygen as terminal electron acceptor. The novel energy-conserving role of H2O2 in this catalase-negative bacterium is discussed in relation to its microaerophilic nature.