A comparison of the heme binding pocket in globins and cytochrome b5.

Of the 85 three-dimensionally characterized residues of cytochrome b5, 51 are found to be structurally and topologically equivalent to the globin fold. When these proteins have been superimposed, the heme irons are found to be less than 1.4 A separated and the heme normals are inclined by less than 9.5 degrees. The proximal histidine of the globins and two adjacent helices are equivalent to the sixth iron ligand and adjacent helices of cytochrome b5. Larger differences in structure are observed on the distal side of the heme, coincident with the most changeable part of the globin structures. The heme itself is rotated by 53 degrees about its normal but such a change is energetically minimal and conservative as the heme side groups are not directly involved in the function of the molecules. The beta-sheet of cytochrome b5 is inserted into a corresponding cavity of the globins forming an additional lining to the heme pocket. The roughly 50 residues missing at the carboxy end of the known cytochrome b5 fragment could correspond in part to the H helix in the globins. While it would seem probable that these similarities represent divergent evolution from a primordial heme-binding protein, the possibility of structural convergence to a functionally satisfactory protein cannot be excluded.     

Characterization of the menaquinone reduction site in the diheme cytochrome b membrane anchor of Wolinella succinogenes NiFe-hydrogenase

The majority of bacterial membrane-bound NiFe-hydrogenases and formate dehydrogenases have homologous membrane-integral cytochrome b subunits. The prototypic NiFe-hydrogenase of Wolinella succinogenes (HydABC complex) catalyzes H2 oxidation by menaquinone during anaerobic respiration and contains a membrane-integral cytochrome b subunit (HydC) that carries the menaquinone reduction site. Using the crystal structure of the homologous FdnI subunit of Escherichia coli formate dehydrogenase-N as a model, the HydC protein was modified to examine residues thought to be involved in menaquinone binding. Variant HydABC complexes were produced in W. succinogenes, and several conserved HydC residues were identified that are essential for growth with H2 as electron donor and for quinone reduction by H2. Modification of HydC with a C-terminal Strep-tag II enabled one-step purification of the HydABC complex by Strep-Tactin affinity chromatography. The tagged HydC, separated from HydAB by isoelectric focusing, was shown to contain 1.9 mol of heme b/mol of HydC demonstrating that HydC ligates both heme b groups. The four histidine residues predicted as axial heme b ligands were individually replaced by alanine in Strep-tagged HydC. Replacement of either histidine ligand of the heme b group proximal to HydAB led to HydABC preparations that contained only one heme b group. This remaining heme b could be completely reduced by quinone supporting the view that the menaquinone reduction site is located near the distal heme b group. The results indicate that both heme b groups are involved in electron transport and that the architecture of the menaquinone reduction site near the cytoplasmic side of the membrane is similar to that proposed for E. coli FdnI.     

Crystal structure of recombinant trypsin-solubilized fragment of cytochrome b(5) and the structural comparison with Val61His mutant

The crystal structure of the recombinant trypsin-solubilized fragment of the microsomal cytochrome b(5) from bovine liver has been determined at 1.9 A resolution and compared with the reported crystal structure of the lipase-solubilized fragment of the membrane protein cytochrome b(5). The two structures are similar to each other. However, some detailed structural differences are observed: the conformation of the segment Asn16-Ser20 is quite different, some helices around the heme and some segments between the helices are shifted slightly, the heme is rotated about the normal of the mean plane of heme, one of the propionates of the heme exhibits a different conformation. The average coordination distances between the iron and the two nitrogen atoms of the imidazole ligands are the same in the two structures. Most of the structural differences can be attributed to the different intermolecular interactions which result from the crystal packing. The wild-type protein structure is also compared with its Val61His mutant, showing that the heme binding and the main chain conformations are basically identical with each other except for the local area of the mutation site. However, when Val61 is mutated to histidine, the large side chain of His61 is forced to point away from the heme pocket toward the solvent region, disturbing the micro-environment of the heme pocket and influencing the stability and the redox potential of the protein.     

Sequence analysis of cytochrome bd oxidase suggests a revised topology for subunit I

Numerous sequences of the cytochrome bd quinol oxidase (cytochrome bd) have recently become available for analysis. The analysis has revealed a small number of conserved residues, a new topology for subunit I and a phylogenetic tree involving extensive horizontal gene transfer. There are 20 conserved residues in subunit I and two in subunit II. Algorithms utilizing multiple sequence alignments predicted a revised topology for cytochrome bd, adding two transmembrane helices to subunit I to the seven that were previously indicated by the analysis of the sequence of the oxidase from E. coli. This revised topology has the effect of relocating the N-terminus and C-terminus to the periplasmic and cytoplasmic sides of the membrane, respectively. The new topology repositions I-H19, the putative ligand for heme b595, close to the periplasmic edge of the membrane, which suggests that the heme b595/heme d active site of the oxidase is located near the outer (periplasmic) surface of the membrane. The most highly conserved region of the sequence of subunit I contains the sequence GRQPW and is located in a predicted periplasmic loop connecting the eighth and ninth transmembrane helices. The potential importance of this region of the protein was previously unsuspected, and it may participate in the binding of either quinol or heme d. There are two very highly conserved glutamates in subunit I, E99 and E107, within the third transmembrane helix (E. coli cytochrome bd-I numbering). It is speculated that these glutamates may be part of a proton channel leading from the cytoplasmic side of the membrane to the heme d oxygen-reactive site, now placed near the periplasmic surface. The revised topology and newly revealed conserved residues provide a clear basis for further experimental tests of these hypotheses. Phylogenetic analysis of the new sequences of cytochrome bd reveals considerable deviation from the 16sRNA tree, suggesting that a large amount of horizontal gene transfer has occurred in the evolution of cytochrome bd.     

Complete amino acid sequence of the cytochrome subunit and amino-terminal sequence of the flavin subunit of flavocytochrome c (sulfide dehydrogenase) from Chlorobium thiosulfatophilum

The complete amino acid sequence of the 86-residue heme subunit of flavocytochrome c (sulfide dehydrogenase) from the green phototrophic bacterium Chlorobium thiosulfatophilum strain Tassajara has been determined as follows: APEQSKSIPRGEILSLSCAGCHGTDGKSESIIPTIYGRSAEYIESALLDFKSGA- RPSTVMGRHAKGYSDEEIHQIAEYFGSLSTMNN. The subunit has a single heme-binding site near the N terminus, consisting of a pair of cysteine residues at positions 18 and 21. The out-of-plane ligands are apparently contributed by histidine 22 and methionine 60. The molecular weight including heme is 10,014. The heme subunit is apparently homologous to small cytochromes c by virtue of the location of the heme-binding site and its extraplanar ligands. However, the amino acid sequence is closer to Paracoccus sp. cytochrome c554(548) (37%) than it is to the heme subunit from Pseudomonas putida p-cresol methylhydroxylase flavocytochrome c (20%). The flavocytochrome c heme subunit is only 14% similar to the small cytochrome c555 also found in Chlorobium. Secondary structure predictions suggest N- and C-terminal helices as expected, but the midsection of the protein probably folds somewhat differently from the small cytochromes of known three-dimensional structure such as Pseudomonas cytochrome c551. Analyses of the residues near the exposed heme edges of the cytochrome subunits of P. putida and C. thiosulfatophilum flavocytochromes c (assuming homology to proteins of known structure) indicate that charged residues are not conserved, suggesting that electrostatic interactions are not involved in the association of the heme and flavin subunits. The N-terminal sequence of the flavoprotein subunit of flavocytochrome has also been determined. It shows no similarity to the comparable region of the p-cresol methylhydroxylase flavoprotein subunit from P. putida. The flavin-binding hexapeptide, isolated and sequenced earlier (Kenney, W. C., McIntire, W., and Yamanaka, T. (1977) Biochim. Biophys. Acta 483, 467-474), is situated at positions 40-46.     

A test of the relationship between sequence and structure in proteins: excision of the heme binding site in apocytochrome b5.

The water-soluble domain of rat hepatic holocytochrome b5 is an alphabeta protein containing elements of secondary structure in the sequence beta1-alpha1-beta4-beta3-alpha2-alpha3-beta5- alpha4-alpha5-beta2-alpha6. The heme group is enclosed by four helices, a2, a3, a4, and a5. To test the hypothesis that a small b hemoprotein can be constructed in two parts, one forming the heme site, the other an organizing scaffold, a protein fragment corresponding to beta1-alpha1-beta4-beta3-lambda-beta2-alpha6 was prepared, where lambda is a seven-residue linker bypassing the heme binding site. The fragment ("abridged b5") was found to contain alpha and beta secondary structure by circular dichroism spectroscopy and tertiary structure by Trp fluorescence emission spectroscopy. NMR data revealed a species with spectral properties similar to those of the full-length apoprotein. This folded form is in slow equilibrium on the chemical shift time scale with other less folded species. Thermal denaturation, as monitored by circular dichroism, absorption, and fluorescence spectroscopy, as well as size-exclusion chromatography-fast protein liquid chromatography (SEC-FPLC), confirmed the coexistence of at least two distinct conformational ensembles. It was concluded that the protein fragment is capable of adopting a specific fold likely related to that of cytochrome b5, but does not achieve high thermodynamic stability and cooperativity. Abridged b5 demonstrates that the spliced sequence contains the information necessary to fold the protein. It suggests that the dominating influence to restrict the conformational space searched by the chain is structural propensities at a local level rather than internal packing. The sequence also holds the properties necessary to generate a barrier to unfolding.     

Structure and reaction mechanisms of multifunctional mitochondrial cytochrome bc1 complex.

The cytochrome bc1 complex from bovine heart mitochondria is a multi-functional enzyme complex. In addition to electron and proton transfer activity, the complex also processes an activatable peptidase activity and a superoxide generating activity. The crystal structure of the complex exists as a closely interacting functional dimer. There are 13 transmembrane helices in each monomer, eight of which belong to cytochrome b, and five of which belong to cytochrome c1, Rieske iron-sulfur protein (ISP), subunits 7, 10 and 11, one each. The distances of 21 A between bL heme and bH heme and of 27 A between bL heme and the iron-sulfur cluster (FeS), accommodate well the observed fast electron transfers between the involved redox centers. However, the distance of 31 A between heme c1 and FeS, makes it difficult to explain the high electron transfer rate between them. 3D structural analyses of the bc1 complexes co-crystallized with the Qu site inhibitors suggest that the extramembrane domain of the ISP may undergo substantial movement during the catalytic cycle of the complex. This suggestion is further supported by the decreased in the cytochrome bc1 complex activity and the increased in activation energy for mutants with increased rigidity in the neck region of ISP.     

Structure of a substrate complex of mammalian cytochrome P450 2C5 at 2.3 A resolution: evidence for multiple substrate binding modes

The structure of rabbit microsomal cytochrome P450 2C5/3LVdH complexed with a substrate, 4-methyl-N-methyl-N-(2-phenyl-2H-pyrazol-3-yl)benzenesulfonamide (DMZ), was determined by X-ray crystallography to 2.3 A resolution. Substrate docking studies and electron density maps indicate that DMZ binds to the enzyme in two antiparallel orientations of the long axis of the substrate. One orientation places the principal site of hydroxylation, the 4-methyl group, 4.4 A from the heme Fe, whereas the alternate conformation positions the second, infrequent site of hydroxylation at >5.9 A from the heme Fe. Comparison of this structure to that obtained previously for the enzyme indicates that the protein closes around the substrate and prevents open access of water from bulk solvent to the heme Fe. This reflects a approximately 1.5 A movement of the F and G helices relative to helix I. The present structure provides a complete model for the protein from residues 27-488 and defines two new helices F' and G'. The G' helix is likely to contribute to interactions of the enzyme with membranes. The relatively large active site, as compared to the volume occupied by the substrate, and the flexibility of the enzyme are likely to underlie the capacity of drug-metabolizing enzymes to metabolize structurally diverse substrates of different sizes.     

Cytochrome c oxidase: evidence for interaction of water molecules with cytochrome a

The resonance Raman spectra of cytochrome c oxidase in protonated buffer compared to that in deuterated buffer indicate that water molecules are near the heme of cytochrome a. Differences in widths of the heme line at 1610 cm-1, after short exposure to D2O, and, additionally, of the heme line at 1625 cm-1, after long exposure, can be accounted for by changes in resonance vibrational energy transfer between modes of cytochrome a2+ and the bending mode of water molecules in the heme pocket. On the basis of the assignment of these modes, we place one water molecule near the vinyl group and one water molecule near the formyl group of the cytochrome a heme. These water molecules may play several possible functional roles.     

On the mode of integration of the thylakoid membrane protein cytochrome b(6) into cytoplasmic membrane of Escherichia coli

In the stroma compartment, several pathways are used for integration/translocation of chloroplast proteins into or across the thylakoid membrane. In this study we investigated the mode of incorporation of the chloroplast-encoded cytochrome b(6) into the bacterial membrane. Cytochrome b(6) naturally comprises of four transmembrane helices (A,B,C,D) and contains two b-type hemes. In the present study, mature cytochrome b(6) or constructed deletion mutants of cytochrome were expressed in E. coli cells. The membrane insertion of cytochrome b(6) in this bacterial model system requires an artificially added presequence that directs the protein to use an E. coli membrane-insertion pathway. This could be accomplished by fusion to maltose-binding protein (MBP) or to the bacterial Sec-dependent signal peptide (SSpelB). The integration of mature cytochrome b(6) into the bacterial cytoplasmic membrane by the Sec pathway has been reported previously by our group (Kroliczewski et al., 2005, Biochemistry, 44: 7570). The results presented here show that cytochrome b(6) devoid of the first helix A can be inserted into the membrane, as can the entire ABCD. On the other hand, the construct devoid of helices A and B is translocated through the membrane into the periplasm without any effective insertion. This suggests the importance of the membrane-anchoring sequences that are likely to be present in only the A and B part, and it is consistent with the results of computational prediction which did not identify any membrane-anchoring sequences for the C or D helices. We also show that the incorporation of hemes into the truncated form of cytochrome b(6) is possible, as long as the B and D helices bearing axial ligands to heme are present.     

Stability and folding kinetics of structurally characterized cytochrome c-b562

The four-helix-bundle protein fold can be constructed from a wide variety of primary amino acid sequences. Proteins with this structure are excellent candidates for investigations of the relationship between folding mechanism and topology. The folding of cytochrome b(562), a four-helix-bundle heme protein, is hampered by heme dissociation. To overcome this complication, we have engineered a variant of cytochrome b(562) (cyt c-b(562)) featuring a c-type linkage between the heme and the polypeptide chain. The replacement of the native cyt b(562) leader sequence in this protein with that of a c-type cytochrome (cyt c(556)) led to high yields of fully matured and correctly folded cyt c-b(562). We have determined the X-ray crystal structure of cyt c-b(562) at 2.25 A and characterized its physical, chemical, and folding properties. These measurements reveal that the c-type linkage does not perturb the protein fold or reduction potential of the heme group. The covalent attachment of the porphyrin to the polypeptide does, however, produce a substantial change in protein stability and folding kinetics.     

Molecular structure of cytochrome c2 isolated from Rhodobacter capsulatus determined at 2.5 A resolution.

The molecular structure of the cytochrome c2, isolated from the purple photosynthetic bacterium Rhodobacter capsulatus, has been solved to a nominal resolution of 2.5 A and refined to a crystallographic R-factor of 16.8% for all observed X-ray data. Crystals used for this investigation belong to the space group R32 with two molecules in the asymmetric unit and unit cell dimensions of a = b = 100.03 A, c = 162.10 A as expressed in the hexagonal setting. An interpretable electron density map calculated at 2.5 A resolution was obtained by the combination of multiple isomorphous replacement with four heavy atom derivatives, molecular averaging and solvent flattening. At this stage of the structural analysis the electron densities corresponding to the side-chains are well ordered except for several surface lysine, glutamate and aspartate residues. Like other c-type cytochromes, the secondary structure of the protein consists of five alpha-helices forming a basket around the heme prosthetic group with one heme edge exposed to the solvent. The overall alpha-carbon trace of the molecule is very similar to that observed for the bacterial cytochrome c2, isolated from Rhodospirillum rubrum, with the exception of a loop, delineated by amino acid residues 21 to 32, that forms a two stranded beta-sheet-like motif in the Rb. capsulatus protein. As observed in the eukaryotic cytochrome c proteins, but not in the cytochrome c2 from Rsp. rubrum, there are two evolutionarily conserved solvent molecules buried within the heme binding pocket.     

Resonance Raman studies of cytochrome P450 2B4 in its interactions with substrates and redox partners

Resonance Raman studies of P450 2B4 are reported for the substrate-free form and when bound to the substrates, benzphetamine (BZ) or butylated hydroxytoluene (BHT), the latter representing a substrate capable of inducing an especially effective conversion to the high-spin state. In addition to studies of the ferric resting state, spectra are acquired for the ferrous CO ligated form. Importantly, for the first time, the RR technique is effectively applied to interrogate the changes in active site structure induced by binding of cytochrome P450 reductase (CPR) and Mn(III) cytochrome b 5 (Mn cyt b 5); the manganese derivative of cyt b 5 was employed to avoid spectroscopic interferences. The results, consistent with early work on mammalian P450s, demonstrate that substrate structure has minimal effects on heme structure or the FeCO fragment of the ferrous CO derivatives. Similarly, the data indicate that the protein is flexible and that substrate binding does not exert significant strain on the heme peripheral groups, in contrast to P450 cam, where substantial effects on heme peripheral groups are seen. However, significant differences are observed in the RR spectra of P450 2B4 when bound with the different redox partners, indicating that the heme structure is clearly sensitive to perturbations near the proximal heme binding site. The most substantial changes are displacements of the peripheral vinyl groups toward planarity with the heme macrocycle by cyt b 5 but away from planarity by CPR. These changes can have an impact on heme reduction potential. Most interestingly, these RR results support an earlier observation that the combination of benzphetamine and cyt b 5 binding produce a synergy leading to unique active site structural changes when both are bound.     

Plasmon waveguide resonance spectroscopic evidence for differential binding of oxidized and reduced Rhodobacter capsulatus cytochrome c2 to the cytochrome bc1 complex mediated by the conformation of the Rieske iron-sulfur protein

The dissociation constants for the binding of Rhodobacter capsulatus cytochrome c2 and its K93P mutant to the cytochrome bc1 complex embedded in a phospholipid bilayer were measured by plasmon waveguide resonance spectroscopy in the presence and absence of the inhibitor stigmatellin. The reduced form of cytochrome c2 strongly binds to reduced cytochrome bc1 (Kd = 0.02 microM) but binds much more weakly to the oxidized form (Kd = 3.1 microM). In contrast, oxidized cytochrome c2 binds to oxidized cytochrome bc1 in a biphasic fashion with Kd values of 0.11 and 0.58 microM. Such a biphasic interaction is consistent with binding to two separate sites or conformations of oxidized cytochrome c2 and/or cytochrome bc1. However, in the presence of stigmatellin, we find that oxidized cytochrome c2 binds to oxidized cytochrome bc1 in a monophasic fashion with high affinity (Kd = 0.06 microM) and reduced cytochrome c2 binds less strongly (Kd = 0.11 microM) but approximately 30-fold more tightly than in the absence of stigmatellin. Structural studies with cytochrome bc1, with and without the inhibitor stigmatellin, have led to the proposal that the Rieske protein is mobile, moving between the cytochrome b and cytochrome c1 components during turnover. In one conformation, the Rieske protein binds near the heme of cytochrome c1, while the cytochrome c2 binding site is also near the cytochrome c1 heme but on the opposite side from the Rieske site, where cytochrome c2 cannot directly interact with Rieske. However, the inhibitor, stigmatellin, freezes the Rieske protein iron-sulfur cluster in a conformation proximal to cytochrome b and distal to cytochrome c1. We conclude from this that the dual conformation of the Rieske protein is primarily responsible for biphasic binding of oxidized cytochrome c2 to cytochrome c1. This optimizes turnover by maximizing binding of the substrate, oxidized cytochrome c2, when the iron-sulfur cluster is proximal to cytochrome b and minimizing binding of the product, reduced cytochrome c2, when it is proximal to cytochrome c1.     

The molecular basis of inhibitor resistance in a mammalian mitochondrial cytochrome b mutant

The mitochondrial gene for the cytochrome b of Complex III has been cloned from a mouse L-cell mutant with increased resistance to 2-n-heptyl-4-hydroxyquinoline-N-oxide and other inhibitors which block reactions at the b562 heme group. Nucleotide sequencing revealed that this gene contained a G:A transition on the coding strand at position 14,830. At the amino acid level, this mutation results in the substitution of an aspartic acid residue for a conserved glycine at position 231 of cytochrome b. Based upon current models for the secondary structure of cytochrome b, the altered amino acid lies in close proximity to one of the invariant histidine residues involved in binding the heme groups. Combining this result with the previous biochemical studies of this mutant, we hypothesize that the insertion of this highly charged side chain alters the conformation around the b562 heme group such that 2-n-heptyl-4-hydroxyquinoline-N-oxide and the other inhibitors of this group have reduced access to the inhibitor binding domain.     

Solution structure of the functional domain of Paracoccus denitrificans cytochrome c552 in the reduced state.

In order to determine the solution structure of Paracoccus denitrificans cytochrome c552 by NMR, we cloned and isotopically labeled a 10.5-kDa soluble fragment (100 residues) containing the functional domain of the 18.2-kDa membrane-bound protein. Using uniformly 15N-enriched samples of cytochrome c552 in the reduced state, a variety of two-dimensional and three-dimensional heteronuclear double-resonance NMR experiments was employed to achieve complete 1H and 15N assignments. A total of 1893 distance restraints was derived from homonuclear 2D-NOESY and heteronuclear 3D-NOESY spectra; 1486 meaningful restraints were used in the structure calculations. After restrained energy minimization a family of 20 structures was obtained with rmsd values of 0.56 +/- 0. 10 A and 1.09 +/- 0.09 A for the backbone and heavy atoms, respectively. The overall topology is similar to that seen in previously reported models of this class of proteins. The global fold consists of two long helices at the N-terminus and C-terminus and three shorter helices surrounding the heme moiety; the helices are connected by well-defined loops. Comparison with the X-ray structure shows some minor differences in the positions of the Trp57 and Phe65 side-chain rings as well as the heme propionate groups.     

Molecular analysis of the cytochrome bc 1-aa 3 branch of the Corynebacterium glutamicum respiratory chain containing an unusual diheme cytochrome c 1

In this work, the genes for cytochrome aa3 oxidase and the cytochrome bc1 complex in the gram-positive soil bacterium Corynebacterium glutamicum were identified. The monocistronic ctaD gene encoded a 65-kDa protein with all features typical for subunit I of cytochrome aa3 oxidases. A ctaD deletion mutant lacked the characteristic 600 nm peak in redox difference spectra, and growth in glucose minimal medium was strongly impaired. The genes encoding subunit III of cytochrome aa3 (ctaE) and the three characteristic subunits of the cytochrome bc1 complex (qcrABC) were clustered in the order ctaE-qcrCAB. Analysis of the deduced primary structures revealed a number of unusual features: (1) cytochrome c1 (QcrC, 30 kDa) contained two Cys-X-X-Cys-His motifs for covalent heme attachment, indicating that it is a diheme c-type cytochrome; (2) the 'Rieske' iron-sulphur protein (QcrA, 45 kDa) contained three putative transmembrane helices in the N-terminal region rather than only one; and (3) cytochrome b (QcrB, 60 kDa) contained, in addition to the conserved part with eight transmembrane helices, a C-terminal extension of about 120 amino acids, which presumably is located in the cytoplasm. Staining of C. glutamicum proteins for covalently bound heme indicated the presence of a single, membrane-bound c-type cytochrome with an apparent molecular mass of about 31 kDa. Since this protein was missing in a qcrCAB deletion mutant, it most likely corresponds to cytochrome c1. Similar to the deltactaD mutant, the deltaqcrCAB mutant showed strongly impaired growth in glucose minimal medium, which indicates that the bc1-aa3 pathway is the main route of respiration under these conditions.     

Cytochrome c reductase purified from Crithidia fasciculata contains an atypical cytochrome c1.

Cytochrome c reductase purified from the trypanosomatid Crithidia fasciculata retained antimycin A sensitivity and catalyzed the reduction of horse heart ferricytochrome c in the presence of reduced coenzyme Q10. The complex contained heme b and heme c1 in a ratio of 2:1. Nine major protein bands ranging in size from 55.3 to approximately 12.8 kDa were resolved by SDS-polyacrylamide gel electrophoresis. A 31.6-kDa protein was identified as cytochrome c1 by the presence of a covalently attached heme. A red shift in the alpha-absorbance band of the cytochrome c1 absolute absorbance spectrum, difference absorbance spectrum, and pyridine ferrohemochrome absorbance spectrum suggested that the heme prosthetic group of C. fasciculata cytochrome c1 is bound to the apoprotein through only one thioether bond. A fragment of the cytochrome c1 gene was amplified from C. fasciculata, Trypanosoma brucei, Leishmania tarentolae, and Bodo caudatus. The deduced heme binding site sequence of each of these kinetoplastid species, Phe-Ala-Pro-Cys-His, contains a phenylalanine rather that a cysteine at the first position so that only one thioether bond can be formed between heme and apoprotein. This phenylalanine substitution and the presence of a conserved proline in the sequence may represent compensatory changes that are necessary for optimal interaction of the cytochromes c1 with the atypical cytochromes c of these species.     

Role of heme in structural organization of cytochrome c probed by semisynthesis

The heme prosthetic group of cytochrome c is covalently attached to the protein through thioether bonds to two cysteine side chains. The role of covalent heme attachment to cytochrome c is not understood, and most heme proteins bind the prosthetic group by iron ion ligation and tertiary interactions only. A two-armed attachment seems redundant if the role of covalent connection is to limit heme group orientation or to decouple heme affinity from redox potential. These considerations suggested that one role for covalent attachment of the rigid planar heme might be in organizing the cytochrome c protein structure. Indeed, porphyrin cytochrome c (in which the heme iron ion has been removed) is substantially more ordered than apocytochrome c, having characteristics consistent with a molten globule state. To assess the importance of planar rigidity in ordering this protein, semisynthesis was used to substitute porphyrin by two hydrophobic surrogates, one based on biphenyl and the other on phenanthrene, which have different degrees of planarity and rigidity. The expected two-armed covalent attachment of each surrogate was confirmed in the protein products by a variety of methods including mass spectrometry and NMR. Despite being only about half the size of the porphyrin macrocycle, and lacking any possibility for ligation or polar group interactions with the surrounding protein, the two surrogates confer helix contents that are comparable to that of the molten globule formed by porphyrin cytochrome c under similar solution conditions. The pH titrations of the derivatives monitored by circular dichroism exhibit reversible, bell-shaped folding and unfolding transitions, implying that charge group interactions in the protein are involved in stabilizing the helical structures formed. The thermal transitions of the two derivatives at neutral pH are cooperative, with similar midpoints. The similarity of helical content and structural stability in the two derivatives indicates that the increase in conformational freedom by the biphenyl surrogate does not substantially reduce protein structural stability. The similarity of the two derivatives to porphyrin cytochrome c suggests that the common feature among the three covalently attached groups-their hydrophobicity-is by far the dominant factor in organizing stable structures in the protein.     

High-resolution refinement of yeast iso-1-cytochrome c and comparisons with other eukaryotic cytochromes c

The structure of yeast iso-1-cytochrome c has been refined against X-ray diffraction data to a nominal resolution of 1.23 A. The atomic model contains 893 protein atoms, as well as 116 water molecules and one sulfate anion. Also included in the refinement are 886 hydrogen atoms belonging to the protein molecule. The crystallographic R-factor is 0.192 for the 12,513 reflections with F greater than or equal to 3 sigma (F) in the resolution range 6.0 to 1.23 A. Co-ordinate accuracy is estimated to be better than 0.18 A. The iso-1-cytochrome c molecule has the typical cytochrome c fold, with the polypeptide chain organized into a series of alpha-helices and reverse turns that serve to envelop the heme prosthetic group in a hydrophobic pocket. Inspection of the conformations of helices in the molecule shows that the local environments of the helices, in particular the presence of intrahelical threonine residues, cause distortions from ideal alpha-helical geometry. Analysis of the internal mobility of iso-1-cytochrome c, based on refined crystallographic temperature factors, shows that the most rigid parts of the molecule are those that are closely associated with the heme group. The degree of saturation of hydrogen-bonding potential is high, with 90% of all polar atoms found to participate in hydrogen bonding. The geometry of intramolecular hydrogen bonds is typical of that observed in other high-resolution protein structures. The 116 water molecules present in the model represent about 41% of those expected to be present in the asymmetric unit. The majority of the water molecules are organized into a small number of hydrogen-bonding networks that are anchored to the protein surface. Comparison of the structure of yeast iso-1-cytochrome c with those of tuna and rice cytochromes c shows that these three molecules have very high structural similarity, with the atomic packing in the heme crevice region being particularly highly conserved. Large conformational differences that are observed between these cytochromes c can be explained by amino acid substitutions. Additional subtle differences in the positioning of the side-chains of several highly conserved residues are also observed and occur due to unique features in the local environments of each cytochrome c molecule.(ABSTRACT TRUNCATED AT 400 WORDS)