Conserved nonplanar heme distortions in cytochromesc

A nonplanar distortion of the heme ofc-type cytochromes is conserved in the proteins isolated from diverse species based upon a comprehensive analysis of available highresolution X-ray crystal structures. This distortion is induced through the cysteine thioether linkages between the porphyrin pyrrole groups and the polypeptide and results in an asymmetric pyrrole distortion. This asymmetry in the heme distortion is also conserved. For other heme proteins which lack these covalent bonds, nearly planar porphyrins are observed. Resonance Raman evidence indicates that nonplanar distortion of porphyrins containing metals, like iron, with large core sizes (?2.00 Å) is energetically unfavorable and can occur only in the presence of significant environmental perturbations. Further, energy minimization and dynamics calculations on the ferric form of yeast iso-1-cytochromec, starting from the crystallographic coordinates and using a molecular mechanics force field which accurately reproduces nonplanar distortions in metalloporphyrins, suggest that this distortion is indeed maintained by the protein tertiary structure. It is proposed that this protein-linked heme distortion modulates electron transfer function through modification of redox potentials of the porphyrin ring and the protein binding properties ofc-type cytochromes.     

During Cytochrome c Maturation CcmI Chaperones the Class I Apocytochromes until the Formation of Their b-Type Cytochrome Intermediates

The c-type cytochromes are electron transfer proteins involved in energy transduction. They have heme-binding (CXXCH) sites that covalently ligate heme b via thioether bonds and are classified into different classes based on their protein folds and the locations and properties of their cofactors. Rhodobacter capsulatus produces various c-type cytochromes using the cytochrome c maturation (Ccm) System I, formed from the CcmABCDEFGHI proteins. CcmI, a component of the heme ligation complex CcmFHI, interacts with the heme-handling protein CcmE and chaperones apocytochrome c₂ by binding its C-terminal helix. Whether CcmI also chaperones other c-type apocytochromes, and the effects of heme on these interactions were unknown previously. Here, we purified different classes of soluble and membrane-bound c-type apocytochromes (class I, c₂ and c₁, and class II c′) and investigated their interactions with CcmI and apoCcmE. We report that, in the absence of heme, CcmI and apoCcmE recognized different classes of c-type apocytochromes with different affinities (nm to μm K_D values). When present, heme induced conformational changes in class I apocytochromes (e.g. c₂) and decreased significantly their high affinity for CcmI. Knowing that CcmI does not interact with mature cytochrome c₂ and that heme converts apocytochrome c₂ into its b-type derivative, these findings indicate that CcmI holds the class I apocytochromes (e.g. c₂) tightly until their noncovalent heme-containing b-type cytochrome-like intermediates are formed. We propose that these intermediates are subsequently converted into mature cytochromes following the covalent ligation of heme via the remaining components of the Ccm complex.     

The biosynthesis of bacterial and plastidic c-type cytochromes

The biosynthesis of bacterial and plastidic c-type cytochromes includes several steps that occur post-translationally. In the case of bacterial cytochromes, the cytosolically synthesized pre-proteins are translocated across the cytoplasmic membrane, the pre-proteins are cleaved to their mature forms and heme is ligated to the processed apoprotein. Although heme attachment has not been studied extensively at the biochemical level, molecular genetic approaches suggest that the reaction generally occurs after translocation of the apoprotein to the periplasm. Recent studies with Bradyrhizobium japonicum and Rhodobacter capsulatus indicate that the process of heme attachment requires the function of a large number of genes. Mutation of these genes generates a pleiotropic deficiency in all c-type cytochromes, suggesting that the gene products participate in processes required for the biosynthesis of all c-type cytochromes. In eukaryotic cells, the biosynthesis of photosynthetic c-type cytochromes is somewhat more complex owing to the additional level of compartmentation. Nevertheless, the basic features of the pathway appear to be conserved. For instance, as is the case in bacteria, translocation and processing of the pre-proteins is not dependent on heme attachment. Genetic analysis suggests that the nuclear as well as the plastid genomes encode functions required for heme attachment, and that these genes function in the biosynthesis of the membrane-associated as well as the soluble c-type cytochrome of chloroplasts. A feature of cytochromes c biogenesis that appears to be conserved between chloroplasts and mitochondria is the sub-cellular location of the heme attachment reaction (p-side of the energy transducing membrane). Continued investigation of all three experimental systems (bacteria, chloroplasts, mitochondria) is likely to lead to a greater understanding of the biochemistry of cytochrome maturation as well as the more general problem of cofactor-protein association during the assembly of an energy transducing membrane.Abbreviations CCHL cytochrome c/heme lyase - CC₁HL cytochrome cl/heme lyase - cyt cytochrome - EMS ethyl methane sulphonate - n-side electrochemically negative side of an energy transducing membrane - p-side electrochemically positive side of an energy transducing membrane - PhoA alkaline phosphatase (encoded by the phoA locus)     

The role ofc-type cytochromes in the terminal respiratory chain of the methylotrophic bacteriumMethylophilus methylotrophus

Whole cells of the methylotrophic bacteriumMethylophilus methylotrophus cultured under methanol-limited conditions contain approximately equal amounts of two majorc-type cytochromes,c _H andc _L. Virtually all of the cytochromec _H, and over one-third of the cytochromec _L, are loosely attached to the periplasmic surface of the respiratory membrane whence they can be released by sonication or by washing cells in ethylenediaminetetraacetate (EDTA). The latter causes inhibition of methanol oxidase activity and stimulation of ascorbate-N,N,N,N-tetramethyl-p-phenylenediamine (TMPD) oxidase activity, neither of which effects are reversible by divalent metal ions. Kinetic analyses indicate that ascorbate-TMPD is oxidised via two routes, viz. a slow low-affinity pathway involving loosely membrane-boundc-type cytochromes plus cytochrome oxidaseaa ₃, and a faster higher-affinity pathway involving the firmly membrane-bound cytochrome oxidasec _L o complex; the former route predominates in the presence of divalent metal ions, and the latter route after exposure to EDTA. These and other results are discussed in terms of the spatial organisation of the terminal respiratory chain, and of the role ofc-type cytochromes in the oxidation of methanol and ascorbate-TMPD.Abbreviations EDTA Enthylenediaminetetraacetate - PMS Phenazinemethosulphate - TMPD N,N,N,N-tetramethyl-p-phenylenediamine - SDS Sodium dodecylsulphate - I₅₀ Concentration of inhibitor required to give 50% inhibition of enzyme activity - PQQ Pyrroloquinoline quinone     

Metabolic pathways inParacoccus denitrificans and closely related bacteria in relation to the phylogeny of prokaryotes

Denitrification and methylotrophy inParacoccus denitrificans are discussed. The properties of the enzymes of denitrification: the nitrate-nitrite antiporter, nitrate reductase, nitrite reductase, nitric oxide reductase and nitrous oxide reductase are described. The genes for none of these proteins have yet been cloned and sequenced fromP. denitrificans. A number of sequences are available for enzymes fromEscherichia coli, Pseudomonas stutzeri andPseudomonas aeruginosa. It is concluded that pathway specificc-type cytochromes are involved in denitrification. At least 40 genes are involved in denitrification.In methanol oxidation at least 20 genes are involved. In this case too pathway specificc-type cytochromes are involved. The sequence homology between the quinoproteins methanol dehydrogenase, alcoholdehydrogenase and glucose dehydrogenase is discussed. This superfamily of proteins is believed to be derived from a common ancestor. ThemoxFJGI operon determines the structural components of methanol dehydrogenase and the associatedc-type cytochrome. Upstream of this operon 3 regulatory proteins were found. The mox Y protein shows the general features of a sensor protein and the moxX protein those of a regulatory protein. Thus a two component regulatory system is involved in both denitrification and methylotrophy.The phylogeny of prokaryotes based on 16S rRNA sequence is discussed. It is remarkable that the 16S rRNA ofThiosphaera pantotropha is identical to that ofP. denitrificans. Still these bacteria show a number of differences.T. pantotropha is able to denitrify under aerobic circumstances and it shows heterotrophic nitrification. Nitrification and heterotrophic nitrification are found in species belonging to the -and -subdivisions of purple non-sulfur bacteria. Thus the occurrence of heterotrophic nitrification inT. pantotropha which belongs to the -subdivision of purple non-sulfur bacteria is a remarkable property. FurthermoreT. pantotropha contains two nitrate reductases of which the periplasmic one is supposed to be involved in aerobic denitrification. The nitrite reductase is of the Cu-type and not of the cytochromecd ₁ type as inP. denitrificans. Also the cytochromeb of theQbc complex ofT. pantotropha is highly similar to its counterpart inP. denitrificans. It is hypothesized that the differences between these two organisms which both contain large megaplasmids is due to a combination of loss of genetic information and plasmid-coded properties. The distribution of a number of complex metabolic systems in eubacteria and in a number of species belonging to the -group of purple non sulphur bacteria is reviewed. Two possibilities to explain this haphazard distribution are considered: 1. Lateral gene transfer between distantly related micro organisms occurs frequently. 2. The eubacterial ancestors must have possessed already these properties. The distribution of these properties is due to sporadic loss during evolutionary divergence.With respect to the occurrence and frequency of lateral gene transfer two opposing views exist. According to molecular biologists lateral gene transfer occurs frequently and is very easy. Bacteria are supposed to form one large gene pool. On the other hand population geneticists have provided evidence that strong systems operate that establish reproductive isolation between diverged species and even between closely related cell lines.Data on amino acid sequences of nitrogenase proteins, cytochromesc, cytochrome oxidases, -subunits of ATP synthase and tryptophan biosynthetic enzymes of various micro organisms were reviewed. In all these cases phylogenetic trees could be constructed based on the amino acid sequence data. In all cases this phylogenetic tree was similar to the one based on 16S rRNA homology. Only in one case evidence for the occurrence of lateral gene transfer was obtained. Therefore it is concluded that lateral gene transfer played a minor role in the distribution of complex metabolic systems among prokaryotes. It must be stressed that this does not exclude the possibility that lateral gene transfer occurred frequently in the initial stage of bacterial evolution. It is hypothesized that the appearance of nitrogen fixation, denitrification and cytochrome oxidase formation were early events in the evolution of micro organisms. Both systems are supposed to have evolved only once. Subsequently the capacity to fix nitrogen or to denitrifymust have been lost many times, just as photosynthetic capacity is supposed to have been lost many times. During evolution many systems have been lost leading to a haphazard distribution of metabolic characters among bacteria. As an example it is suggested that organisms with a respiratory chain similar to that ofEscherichia coli arose by loss of the capacity to form the Qbc complex andc-type cytochromes. The remaining systems could be controlled much better however than in the ancestral organisms.     

Defects in cytochrome cd 1-dependent nitrite respiration of transposon Tn5-induced mutants from Pseudomonas stutzeri

Mutants with defective respiratory nitrite utilization (Nir^- phenotype) were obtained by transposon Tn5 insertion into genomic DNA of the ZoBell strain of Pseudomonas stutzeri. Three representative mutants were characterized with respect to their activities of nitrite and nitric oxide reduction, cytochrome cd ₁ content, and pattern of soluble c-type cytochromes. Mutant strain MK201 over-produced cytochrome c ₅₅₂ about fourfold by comparison with the wild type, but possessed an in vitro functional cytochrome cd ₁. Mutant strain MK202 lacked cytochrome cd ₁ and, simultaneously, had low amounts of cytochrome c ₅₅₂ and the split -peak c-type cytochrome. Strain MK203 synthesized nitrite reductase defective in the heme d ₁ prosthetic group. Irrespective of these biochemically distinct Nir^- phenotypes, all mutants preserved the nitric oxidereducing capability of the wild type. The mutant characteristics demonstrate that cytochrome cd ₁ is essential for nitrite respiration of P. stutzeri and establish the presence of a nitric oxide-reducing system distinct from cytochrome cd ₁. They also indicate the functional or regulatory interdependence of c-type cytochromes.     

The role of the genesnrf EFG andccmFH in cytochromec biosynthesis inEscherichia coli

It has been suggested that two groups ofEscherichia coli genes, theccm genes located in the 47-min region and thenrfEFG genes in the 92-min region of the chromosome, are involved in cytochromec biosynthesis during anaerobic growth. The involvement of the products of these genes in cytochromec synthesis, assembly and secretion has now been investigated. Despite their similarity to other bacterial cytochromec assembly proteins, NrfE, F and G were found not to be required for the biosynthesis of any of thec-type cytochromes inE. coli. Furthermore, these proteins were not required for the secretion of the periplasmic cytochromes, cytochromec ₅₅₀ and cytochromec ₅₅₂, or for the correct targeting of the NapC and NrfB cytochromes to the cytoplasmic membrane. NrfE and NrfG are required for formate-dependent nitrite reduction (the Nrf pathway), which involves at least twoc-type cytochromes, cytochromec ₅₅₂ and NrfB, but NrfF is not essential for this pathway. Genes similar tonrfE, nrfF andnrfG are present in theE. coli nap-ccm locus at minute 47. CcmF is similar to NrfE, the N-terminal region of CcmH is similar to NrfF and the C-terminal portion of CcmH is similar to NrfG. In contrast to NrfF, the N-terminal, NrfF-like portion of CcmH is essential for the synthesis of allc-type cytochromes. Conversely, the NrfG-like C-terminal region of CcmH is not essential for cytochromec biosynthesis. The data are consistent with proposals from this and other laboratories that CcmF and CcmH form part of a haem lyase complex required to attach haemc to C-X-X-C-H haem-binding domains. In contrast, NrfE and NrfG are proposed to fulfill a more specialised role in the assembly of the formate-dependent nitrite reductase.     

Formation of active nitrite reductase (cytochromecd 1) in the strainParacoccus denitrificans HUUG25

Strain HUUG25 ofParacoccus denitrificans has been frequently thought to be devoid of allc-type cytochromes. We show here by means of enzymological and immunological techniques that the mutant synthesizes active nitrite reductase (cytochromecd ₁) upon prolonged exposure to microoxic conditions. The synthesis occurred faster in the presence of exogenous hemin. The time pattern of 5-aminolevulinate synthase activity was also altered by the mutation. These findings suggest a defective regulation of heme supply to the site of nitrite reductase assembly in the periplasm.     

Spectral characterization of c-type cytochromes purified from Beggiatoa alba

Three c-type cytochromes were purified from the filamentous sulfur-oxidizing bacterium, Beggiatoa alba strain B18LD, by ammonium sulfate fractionation, flat bed isoelectric focusing and gel filtration. Two of the cytochromes; flavocytochrome c-554 and cytochrome c, were similar to cytochromes found in anoxygenic photosynthetic bacteria. Flavocytochrome c-554 had an apparent molecular weight of 21,000, an isoelectric focusing point at pH 4.4, contained FMN as the flavin component and had absorption maxima at 410, 450 and 470 nm in the oxidized form and at 417, 523 and 554 nm in the dithionite-reduced from. Cytochrome c was also an acidic protein with a pI of 4.8 and an apparent molecular weight of 18,000. The absorption spectra maxima were at 400, 490 and 635 nm in the oxidized form, at 424 and 550 nm in the dithione-reduced form and at 415 and 555 nm in the dithionite-reduced plus CO form. The third cytochrome characterized, cytochrome c-553 had an apparent molecular weight of 13,000, an isoelectric point at pH 4.4 and showed absorption maxima at 411 nm in the oxidized form and at 418, 523 and 553 nm in the dithionite-reduced form. Cytochrome c-553 was also isolated as a complex with a non-heme protein with a molecular weight of 16,000. The non-heme protein altered the absorption spectra and isoelectric point of cytochrome c-553.Abbreviations IEF isoelectric focusing - M _r molecular weight - pI isoelectric point     

Biogenesis of mitochondrialc-type cytochromes

Cytochromesc andc ₁ are essential components of the mitochondrial respiratory chain. In both cytochromes the heme group is covalently linked to the polypeptide chain via thioether bridges. The location of the two cytochromes is in the intermembrane space; cytochromec is loosely attached to the surface of the inner mitochondrial membrane, whereas cytochromec ₁ is firmly anchored to the inner membrane. Both cytochromec andc ₁ are encoded by nuclear genes, translated on cytoplasmic ribosomes, and are transported into the mitochondria where they become covalently modified and assembled. Despite the many similarities, the import pathways of cytochromec andc ₁ are drastically different. Cytochromec ₁ is made as a precursor with a complex bipartite presequence. In a first step the precursor is directed across outer and inner membranes to the matrix compartment of the mitochondria where cleavage of the first part of the presequence takes place. In a following step the intermediate-size form is redirected across the inner membrane; heme addition then occurs on the surface of the inner membrane followed by the second processing reaction. The import pathway of cytochromec is exceptional in practically all aspects, in comparison with the general import pathway into mitochondria. Cytochromec is synthesized as apocytochromec without any additional sequence. It is translocated selectively across the outer membrane. Addition of the heme group, catalyzed by cytochromec heme lyase, is a requirement for transport. In summary, cytochromec ₁ import appears to follow a conservative pathway reflecting features of cytochromec ₁ sorting in prokaryotic cells. In contrast, cytochromec has invented a rather unique pathway which is essentially non-conservative.     

Electron transport and respiration in Beggiatoa and Vitreoscilla

Seven strains of bacteria belonging to the Beggiatoa-Vitreoscilla group were studied for their respiratory activity and for the presence of electron transport conponents. All strains tested oxidized [1-¹⁴C] and [2-¹⁴C] acetate to ¹⁴CO₂ at relatively high rates. All strains tested were N,N,N,N-tetramethylphenylenediamine (TMPD)-oxidase positive and contained spectra representing a-type and carbon monoxide-binding cytochromes. Most of the strains also contained spectra representing c-type and b-type cytochromes. Beggiatoa alba B18LD contained b-type, a-type, c-type and CO-binding cytochromes, the latter two being located in the 144,000 x g soluble fraction. B. alba also contained ubiquinone-8 as its only detectable quinone.Non-standard abbreviations BSS basal salts solution - BH Beggiatoa heterotrophic medium - BSO Beggiatoa sulfide oxidation medium - TMPD N,N,N,N-tetramethylphenylenediamine - Q8 ubiquinone-8     

Cytochromes of the non-thiosulfate-utilizing green sulfur bacterium Chlorobium limicola

Flavocytochrome c-553 of the non-thiosulfateutilizing green sulfur bacterium Chlorobium limicola strain 6330 was partially purified by ion exchange column chromatography and ammonium sulfate fractionation (highest purity index obtained: A ₂₈₀/A _{417 red}=0.96). It is autoxidizable and located in the soluble fraction. This hemoprotein contains a flavin component and one heme per molecule. The dithionite reduced spectrum reveals the typical maxima of a c-type cytochrome: =553,5 nm; =523 nm; =417 nm, while the oxidized form shows a -band at 410 nm and two shoulders at 440 nm and 480 nm indicating the flavin component. The flavocytochrome is a basic protein with an isoelectric point at pH 9.0 (± 0.5), a redox potential of 65 mV, a molecular weight of 56,000. It participates in sulfide oxidation and shows neither adenylylsulfate reductase nor sulfite reductase activity. C. limicola further contains a soluble cytochrome c-555 (highest purity index obtained: A ₂₈₀/A _{412 ox}=0.13; isoelectric point between pH 9.5 and 10) and the non-heme iron-containing proteins rubredoxin and ferredoxin, but lacks cytochrome c-551. Besides these soluble electron transfer proteins a membrane-bound c-type cytochrome (=554,5 nm) can be detected spectrophotometrically.Non-common abbreviations HIPIP high-potential iron sulfur protein - APS adenylylsulfate     

Porphyrin interactions with wild-type and mutant mouse ferrochelatase

Ferrochelatase (EC 4.99.1.1), the terminal enzyme of the heme biosynthetic pathway, catalyzes Fe(2+) chelation into protoporphyrin IX. Resonance Raman and UV-vis absorption spectroscopies of wild-type and engineered variants of murine ferrochelatase were used to examine the proposed structural mechanism for iron insertion into porphyrin. The recombinant variants (i.e., H207N and E287Q) are enzymes in which the conserved amino acids histidine-207 and glutamate-287 of murine ferrochelatase were substituted with asparagine and glutamine, respectively. Both of these residues are at the active site of the enzyme as deduced from the Bacillus subtilis ferrochelatase three-dimensional structure. On the basis of changes in the UV-vis absorption spectrum, addition of free-base or metalated porphyrins to wild-type ferrochelatase and H207N variant yields a 1:1 complex, most likely a monomeric protein-bound species at the active site. In contrast, the addition of porphyrin (either free base or metalated) to E287Q is substoichiometric, as this variant retains bound porphyrin in the active site during isolation and purification. The specificity of porphyrin binding is confirmed by the narrowing of the structure-sensitive lines and the vinyl vibrational mode in the resonance Raman spectra. Shifts in the resonance Raman lines of free-base and metalated porphyrins bound to the wild-type ferrochelatase indicate a nonplanar distortion of the porphyrin macrocycle. However, the magnitude of the distortion cannot be determined without first defining the specific type of deformation. Significantly, the extent of the nonplanar distortion varies in the case of H207N- and E287Q-bound porphyrins. In fact, resonance Raman spectral decompositions indicate a homogeneous ruffled deformation for the nickel protoporphyrin bound to the wild-type ferrochelatase, whereas both planar and ruffled conformations are present for the H207N-bound porphyrin. Perhaps more revealing is the unusual resonance Raman spectrum of the endogenous E287Q-bound porphyrin, which has the structure-sensitive lines greatly upshifted relative to those of the free-base protoporphyrin in solution. This could be interpreted as an equilibrium between protein conformers, one of which favors a highly distorted porphyrin macrocycle. Taken together, these findings suggest that distortion occurs in murine ferrochelatase for some porphyrins, even without metal binding, which is apparently required for the yeast ferrochelatase.     

Characterization of the Escherichia coli CcmH protein reveals new insights into the redox pathway required for cytochrome c maturation

The CcmH protein of Escherichia coli is encoded by the last gene of the ccm gene cluster required for cytochrome c maturation. A mutant in which the entire ccmH gene was deleted failed to synthesize both indigenous and foreign c-type cytochromes. However, deletion of the C-terminal hydrophilic domain homologous to CycH of other gram-negative bacteria affected neither the biogenesis of indigenous c-type cytochromes nor that of the Bradyrhizobium japonicum cytochrome c ₅₅₀. This confirmed that only the N-terminal domain containing a conserved CXXC motif is required in E. coli. PhoA fusion analysis showed that this domain is periplasmic. Site-directed mutagenesis of the cysteines of the CXXC motif revealed that both cysteines are required for cytochrome c maturation during aerobic growth, whereas only the second cysteine is required for cytochrome c maturation during anaerobic growth. The deficiency of the point mutants was complemented when 2-mercapto-ethanesulfonic acid was added to growing cells; other thiol compounds did not stimulate cytochrome c formation in these strains. We propose a model for the reaction sequence in which CcmH keeps the heme binding site of apocytochrome c in a reduced form for subsequent heme ligation. Received: 7 September 1998 / Accepted: 15 November 1998     

Changes in cytochrome levels during growth of the yeast-like fungusAureobasidium pullulans

Cytochromes ofAureobasidium pullulans have been identified and partially characterized using low-temperature and carbon-monoxide-difference spectroscopy. The presence ofa-,b-, andc-type cytochromes is demonstrated, as are other unidentified redox components. During exponential growth in batch culture, cytochrome levels showed complex changes. Changes in respiration rates and in the levels of cytochromea+a ₃ closely paralleled cellular growth: both increased exponentially until stationary phase, when no further increase occurred. Theb- andc-type cytochromes showed biphasic increases, initially doubling every, generation time and then increasing more slowly during the stationary phase. Sensitivity of respiration to 100M potassium cyanide gradually decreased during exponential growth, falling from virtually 100% inhibition after about 20 h growth to 30% inhibition in the stationary phase. The results suggest that in stationary-phase cultures, an alternative cyanide-insensitive but salicylhydroxamic-acid-sensitive terminal oxidase also operates.     

Horse heart ferricytochrome c: conformation and heme configuration of low ionic strength acidic forms

Resonance Raman, absorption and circular dichroism spectroscopic studies of the stable forms of horse heart ferricytochromec in thepH range 6–0.8 and at the lowest possible ionic strengths, in water, and at 30°C are reported. The neutralpH form, state III, changes to the acidicpH form, state I, through a three-step process: state III state IIIa state II state I, with pKa's of 3.6±0.3, 2.7±0.2, and 1.2±0.2, depending on the monitoring probe, respectively. State IIIa ferricytochromec is like state III (i.e., with the Met-80-sulfur-iron linkage and a closed heme crevice) but with a higher degree of folding and a slightly larger porphyrin core. State II ferricytochromec is an unfolded form with an open heme crevice and no Met-80-sulfur-iron linkage. The heme iron is high-spin and hexacoordinated with weak ligand-field groups, water, and nitrogen of the protonated/hydrogen-bonded imidazole of the His-18 residue at the axial positions. The state I form also lacks the Met-80-sulfur-iron linkage and has an open heme crevice like the state II form; however, it is less unfolded and has a high-spin pentacoordinated heme iron, with the nitrogen of the imidazole of His-18 as the axial ligate, which is out of the porphyrin plane by about 0.45 Å.     

Cytochromes in Thiobacillus tepidarius and the respiratory chain involved in the oxidation of thiosulphate and tetrathionate

Thiobacillus tepidarius was shown to contain cytochrome(s) c with absorption maxima at 421, 522 and 552 nm in room temperature reduced minus oxidized difference spectra, present at 1.1–1.2 nmol per mg dry wt and present in both membrane and soluble fractions of the cell. The membrane-bound cytochrome c (1.75 nmol per mg membrane protein) had a midpoint potential (E_m, pH 7.0) of 337 mV, while the soluble fractions appeared to contain cytochrome(s) c with E_m (pH 7.0) values of about 270 and 360 mV. The organism also contained three distinct membrane-bound b-type cytochromes (totalling 0.33 nmol per mg membrane protein), each with absorption maxima in reduced minus oxidized difference spectra at about 428, 532 and 561 nm. The E_m (pH 7.0) values for the three cytochromes b were 8 mV (47.8% of total), 182 mV (13.7%) and 322 mV (38.5%). No a- or d-type cytochromes were detectable spectrophotometrically in the intact organism or its membrane and soluble fractions. Evidence is presented for both CO-binding and CO-unreactive cytochromes b or o, and CO-binding cytochrome(s) c. From redox effects observed with CO it is proposed that a cytochrome c donates electrons to a cytochrome b, and that a high potential cytochrome b or o may be acting as the terminal oxidase in substrate oxidation. This may be the 445 nm pigment, a photodissociable CO-binding membrane haemoprotein. Substrate oxidation was relatively insensitive to CO-inhibition, but strongly inhibited by cyanide and azide. Thiosulphate oxidation couples directly to cytochrome c reduction, but tetrathionate oxidation is linked (probably via ubiquinone Q-8) to reduction of a cytochrome b of lower potential than the cytochrome c. The nature of possible electron transport pathways in Thiobacillus tepidarius is discussed. One speculative sequence is: c^– b₈ b₁₈₂ c₂₇₀ c₃₃₇ b₃₂₂/c₃₆₀ O₂ Abbreviations E_m midpoint electrode potential - E _inf0 ^sup pH 7, standard electrode potential at pH 7.0 - Q-8 coenzyme Q-8 (ubiquinone-40)     

Effect of oxygen limitation on the formation of the electron transport system of the phytopathogenic fluorescent bacteriumPseudomonas cichorii

The composition of the membrane-bound electron transport system of the phytopathogenic bacteriumPseudomonas cichorii underwent modification in response to oxygen supply. Growth adaptation to low oxygen concentrations was characterized by repression of cytochromes involved in ubiquinol-cyt.c oxidoreductase and cyt.c oxidase activities. By contrast, cyto.o, i.e., the alternative cyanide-insensitive oxidase ofP. cichorii, was unaffected by low oxygen tension. Noa-type cytochromes could be detected at any stage of growth.     

Cytochrome aa 3 from Methylococcus capsulatus (Bath)

A cytochrome aa ₃-type oxidase was isolated with and without a c-type cytochrome (cytochrome c-557) from Methylococcus capsulatus Bath by ion-exchange and hydrophobic chromatography in the presence of Triton X-100. Although cytochrome c-557 was not a constitutive component of the terminal oxidase, the cytochrome c ascorbate-TMPD oxidase activity of the enzyme decreased dramatically when the ratio of cytochrome c-557 to heme a dropped below 1:3. On denaturing gels, the purified enzyme dissociated into three subunits with molecular weights of 46,000, 28,000 and 20,000. The enzyme contains two heme groups (a and a ₃), absorption maximum at 422 nm in the resting state, at 445 and 601 nm in the dithionite reduced form and at 434 and 598 nm in the dithionite reduced plus CO form. Denaturing gels of the cytochrome aa ₃-cytochrome c-557 complex showed the polypeptides associated with cytochrome aa ₃ plus a heme c-staining subunit with a molecular weight of 37,000. The complex contains approximately two heme a, one heme c, absorption maximum at 420 nm in the resting state and at 421, 445, 522, 557 and 601 nm in the dithionite reduced form. The specific activity of the purified enzyme was 130 mol O₂/min · mol heme a compared to 753 mol O₂/min · mol heme a when isolated with cytochrome c-557.Abbreviations MMO methan monooxygenase - sMMO soluble methane monooxygenase - pMMO particulate methane monooxygenase - TMPD N,N,N,N-tetramethyl-p-phenylenediamine dihydrochloride - Na₂EDTA disodium ethylenediamine-tetraacetic acid