Structural and biochemical characterization of DHC2, a novel diheme cytochrome c from Geobacter sulfurreducens

Multiheme cytochromes c constitute a widespread class of proteins with essential functions in electron transfer and enzymatic catalysis. Their functional properties are in part determined by the relative arrangement of multiple heme cofactors, which in many cases have been found to pack in conserved interaction motifs. Understanding the significance of these motifs is crucial for the elucidation of the highly optimized properties of multiheme cytochromes c, but their spectroscopic investigation is often hindered by the large number and efficient coupling of the individual centers and the limited availability of recombinant protein material. We have identified a diheme cytochrome c, DHC2, from the metal-reducing soil bacterium Geobacter sulfurreducens and determined its crystal structure by the method of multiple-wavelength anomalous dispersion (MAD). The two heme groups of DHC2 pack into one of the typical heme interaction motifs observed in larger multiheme cytochromes, but because of the absence of further, interfering cofactors, the properties of this heme packing motif can be conveniently studied in detail. Spectroscopic properties (UV-vis and EPR) of the protein are typical for cytochromes containing low-spin Fe(III) centers with bis-histidinyl coordination. Midpoint potentials for the two heme groups have been determined to be -135 and -289 mV by potentiometric redox titrations. DHC2 has been produced by recombinant expression in Escherichia coli using the accessory plasmid pEC86 and is therefore accessible for systematic mutational studies in further investigating the properties of heme packing interactions in cytochromes c.     

The binding domain on horse cytochrome c and Rhodobacter sphaeroides cytochrome c2 for the Rhodobacter sphaeroides cytochrome bc1 complex

The interaction of the Rhodobacter sphaeroides cytochrome bc1 complex with Rb. sphaeroides cytochrome c2 and horse cytochrome c was studied by using specific lysine modification and ionic strength dependence methods. The rate of the reactions with both cytochrome c and cytochrome c2 decreased rapidly with increasing ionic strength above 0.2 M NaCl. The ionic strength dependence suggested that electrostatic interactions were equally important to the reactions of the two cytochromes, even though they have opposite net charges at pH 7.0. In order to define the interaction domain on horse cytochrome c, the reaction rates of derivatives modified at single lysine amino groups with trifluoroacetyl or trifluoromethylphenylcarbamoyl were measured. Modification of lysine-8, -13, -27, -72, -79, and -87 surrounding the heme crevice was found to significantly lower the rate of the reaction, while modification of lysines in other regions had no effect. This result indicates that lysines surrounding the heme crevice of horse cytochrome c are involved in electrostatic interactions with carboxylate groups at the binding site on the cytochrome bc1 complex. In order to define the reaction domain on cytochrome c2, a fraction consisting of a mixture of singly labeled 4-carboxy-2,6-dinitrophenylcytochrome c2 derivatives modified at lysine-35, -88, -95, -97, and -105 and several unidentified lysines was prepared. Although it was not possible to resolve these derivatives, all of the identified lysines are located on the front surface of cytochrome c2 near the heme crevice. The rate of reaction of this fraction was significantly smaller than that of native cytochrome c2, suggesting that the binding domain on cytochrome c2 is also located at the heme crevice.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of Rhodobacter sphaeroides cytochrome c(2) proteins with altered heme attachment sites

In c-type cytochromes, heme is attached to the polypeptide via thioether linkages between vinyl groups on the tetrapyrrole ring and cysteine thiols in a CX(2)CH motif. To study the role of the heme-binding site in c-type cytochrome assembly and function, we generated amino acid changes in this region of Rhodobacter sphaeroides cytochrome c(2) ((15)Cys-Gln-Thr-Cys-His(19)). Amino acid substitutions at Cys(15), Cys(18), or His(19) produced mutant proteins that did not support growth via photosynthesis where this electron carrier is required. Many of these changes appeared to slow signal peptide removal, suggesting that heme attachment is coupled to processing of the c-type cytochrome precursor protein. Inserting an alanine between the cysteine ligands (CycA-Ins17A) did not significantly alter the behavior of this protein in vivo and in vitro, suggesting that the existence of 2 residues between cysteine thiols is not essential for heme attachment to a Class I c-type cytochrome like cytochrome c(2).     

Regions of Rhodobacter sphaeroides cytochrome c2 required for export, heme attachment, and function.

Cytochrome c2 is a periplasmic redox protein involved in both the aerobic and photosynthetic electron transport chains of Rhodobacter sphaeroides. The process of cytochrome c2 maturation has been analyzed in order to understand the protein sequences involved in attachment of the essential heme moiety to the cytochrome c2 polypeptide and localization of the protein to the periplasm. To accomplish this, five different translational fusions which differ only in the cytochrome c2 fusion junction were constructed between cytochrome c2 and the Escherichia coli periplasmic alkaline phosphatase. All five of the fusion proteins are exported to the periplasmic space. The four fusion proteins that contain the NH2-terminal site of covalent heme attachment to cytochrome c2 are substrates for heme binding, suggesting that the COOH-terminal region of the protein is not required for heme attachment. Three of these hybrids possess heme peroxidase activity, which indicates that they are functional as electron carriers. Biological activity is possessed by one hybrid protein constructed five amino acids before the cytochrome c2 COOH terminus, since synthesis of this protein restores photosynthetic growth to a photosynthetically incompetent cytochrome c2-deficient derivative of R. sphaeroides. Biochemical analysis of these hybrids has confirmed CycA polypeptide sequences sufficient for export of the protein (A. R. Varga and S. Kaplan, J. Bacteriol. 171:5830-5839, 1989), and it has allowed us to identify regions of the protein sufficient for covalent heme attachment, heme peroxidase activity, docking to membrane-bound redox partners, or the capability to function as an electron carrier.     

Cytochrome c" from the obligate methylotroph Methylophilus methylotrophus, an unexpected homolog of sphaeroides heme protein from the phototroph Rhodobacter sphaeroides.

The complete primary structure of an unusual soluble cytochrome c isolated from the obligate methylotrophic bacterium Methylophilus methylotrophus has been determined to contain 124 amino acids and to have an average molecular mass of 14293.0 Da. The sequence has two unusual features: firstly, the location of the heme-binding cysteines is far downstream from the N-terminus, namely at positions 49 and 52; secondly, an extra pair of cysteine residues is present near the C-terminus. In both respects, cytochrome c" is similar to the oxygen-binding heme protein SHP from the purple phototrophic bacterium Rhodobacter sphaeroides. In contrast to SHP, cytochrome c" changes from low-spin to high-spin upon reduction, due to dissociation of a sixth heme ligand histidine which is identified as His-95 by analogy to the class I cytochromes c. The distance of His-95 from the heme (41 residues) and the presence of certain consensus residues suggests that cytochrome c" is the second example of a variant class I cytochrome c.     

Interactions between cytochrome c2 and the photosynthetic reaction center from Rhodobacter sphaeroides: the cation-pi interaction

The cation-pi interaction between positively charged and aromatic groups is a common feature of many proteins and protein complexes. The structure of the complex between cytochrome c(2) (cyt c(2)) and the photosynthetic reaction center (RC) from Rhodobacter sphaeroides exhibits a cation-pi complex formed between Arg-C32 on cyt c(2) and Tyr-M295 on the RC [Axelrod, H. L., et al. (2002) J. Mol. Biol. 319, 501-515]. The importance of the cation-pi interaction for binding and electron transfer was studied by mutating Tyr-M295 and Arg-C32. The first- and second-order rates for electron transfer were not affected by mutating Tyr-M295 to Ala, indicating that the cation-pi complex does not greatly affect the association process or structure of the state active in electron transfer. The dissociation constant K(D) showed a greater increase when Try-M295 was replaced with nonaromatic Ala (3-fold) as opposed to aromatic Phe (1.2-fold), which is characteristic of a cation-pi interaction. Replacement of Arg-C32 with Ala increased K(D) (80-fold) largely due to removal of electrostatic interactions with negatively charged residues on the RC. Replacement with Lys increased K(D) (6-fold), indicating that Lys does not form a cation-pi complex. This specificity for Arg may be due to a solvation effect. Double mutant analysis indicates an interaction energy between Tyr-M295 and Arg-C32 of approximately -24 meV (-0.6 kcal/mol). This energy is surprisingly small considering the widespread occurrence of cation-pi complexes and may be due to the tradeoff between the favorable cation-pi binding energy and the unfavorable desolvation energy needed to bury Arg-C32 in the short-range contact region between the two proteins.     

Flash-photolysis of fully reduced and mixed-valence CO-bound Rhodobacter sphaeroides cytochrome c oxidase: heme spectral shifts

Conformational changes, internal electron transfer, and CO rebinding processes in cytochrome c oxidase from Rhodobacter sphaeroides reduced to different degrees were investigated. The reactions were followed using a gated optical spectrometric multichannel analyzer. Light-induced difference spectra, recorded in the 350-700 nm region over the 100 ns to 1 s time interval, were analyzed by singular value decomposition and global exponential fitting. The photolyzed fully reduced enzyme showed two relaxations, approximately 1 and 190 mus, prior to the 20 ms CO rebinding process. Intramolecular electron transfer was monitored following photolysis of the mixed-valence CO-bound enzyme. The analysis revealed 1.1 micros, 2.4 micros, 31 micros, 68 ms, and 240 ms apparent lifetimes, the first three of which are attributed to electron transfer from heme a3 to heme a with contribution from a relaxation process at the heme a3 site. Spectral changes associated with the microsecond processes are consistent with 75% electron transfer from heme a3 to heme a. A comparison of the experimental spectra and model difference spectra for the intramolecular electron transfer indicated approximately 3 nm blue shift in the absolute spectra of both the oxidized heme a3 and reduced heme a generated in the process. The 68 and 240 ms lifetimes are due to CO recombination to heme a3 and are attributed to the presence of two conformers, the slower rate corresponding to the conformer in higher abundance. The dependency of the apparent rate of CO rebinding on the intensity of the probe beam in single-wavelength experiments is explained.     

The Rhodobacter sphaeroides cytochrome c2 signal peptide is not necessary for export and heme attachment.

Rhodobacter sphaeroides cytochrome c2 (cyt c2) is a member of the heme-containing cytochrome c protein family that is found in the periplasmic space of this gram-negative bacterium. This exported polypeptide is made as a higher-molecular-weight precursor with a typical procaryotic signal peptide. Therefore, cyt c2 maturation is normally expected to involve precursor translocation across the cytoplasmic membrane, cleavage of the signal peptide, and covalent heme attachment. Surprisingly, synthesis as a precursor polypeptide is not a prerequisite for cyt c2 maturation because deleting the entire signal peptide does not prevent export, heme attachment, or function. Although cytochrome levels were reduced about threefold in cells containing this mutant protein, steady-state cyt c2 levels were significantly higher than those of other exported bacterial polypeptides which contain analogous signal peptide deletions. Thus, this mutant protein has the unique ability to be translocated across the cytoplasmic membrane in the absence of a signal peptide. The covalent association of heme with this mutant protein also suggests that the signal peptide is not required for ligand attachment to the polypeptide chain. These results have uncovered some novel aspects of bacterial c-type cytochrome biosynthesis.     

Structural gene of cytochrome b-562 from the cytochrome b-c1 complex of Rhodobacter sphaeroides

The structural gene coding for cytochrome b-562 isolated from the cytochrome b-c1 complex of Rhodobacter (Rhodopseudomonas) sphaeroides has been cloned. Its nucleotide sequence has been determined and the amino acid sequence was deduced therefrom. It consists of 157 amino acids (Mr 17,237) and contains four hydrophobic segments. The first 30 residues in the predicted amino acid sequence are the same as those determined for the NH2-terminal portion of purified cytochrome b-562. The amino acid composition is in accord with that determined for the pure protein. From the hydropathy profile and molar ratio of protoheme to cytochrome b-562, it is suggested that the structural and functional unit of the cytochrome is a two-heme cross-linked homodimer.     

Definition of the interaction domain for cytochrome c on cytochrome c oxidase. Ii. Rapid kinetic analysis of electron transfer from cytochrome c to Rhodobacter sphaeroides cytochrome oxidase surface mutants

The reaction between cytochrome c (Cc) and Rhodobacter sphaeroides cytochrome c oxidase (CcO) was studied using a cytochrome c derivative labeled with ruthenium trisbipyridine at lysine 55 (Ru-55-Cc). Flash photolysis of a 1:1 complex between Ru-55-Cc and CcO at low ionic strength results in electron transfer from photoreduced heme c to Cu(A) with an intracomplex rate constant of k(a) = 4 x 10(4) s(-1), followed by electron transfer from Cu(A) to heme a with a rate constant of k(b) = 9 x 10(4) s(-1). The effects of CcO surface mutations on the kinetics follow the order D214N > E157Q > E148Q > D195N > D151N/E152Q approximately D188N/E189Q approximately wild type, indicating that the acidic residues Asp(214), Glu(157), Glu(148), and Asp(195) on subunit II interact electrostatically with the lysines surrounding the heme crevice of Cc. Mutating the highly conserved tryptophan residue, Trp(143), to Phe or Ala decreased the intracomplex electron transfer rate constant k(a) by 450- and 1200-fold, respectively, without affecting the dissociation constant K(D). It therefore appears that the indole ring of Trp(143) mediates electron transfer from the heme group of Cc to Cu(A). These results are consistent with steady-state kinetic results (Zhen, Y., Hoganson, C. W., Babcock, G. T., and Ferguson-Miller, S. (1999) J. Biol. Chem. 274, 38032-38041) and a computational docking analysis (Roberts, V. A., and Pique, M. E. (1999) J. Biol. Chem. 274, 38051-38060).     

The interaction between heme and protein in cytochrome c1

The hormonal regulation of two regulatory enzymes of fatty acid synthesis acetyl-CoA carboxylase (EC 6.4.1.2) and glucose-6-phosphate dehydrogenase (EC 1.1.1.49), has been investigated in human diploid fibroblasts. There was a 35% increase in acetyl-CoA carboxylase activity, 72 h following addition of 10 microU/ml insulin to the culture medium. Addition of 1 microgram/ml of 3,3'5-triiodothyronine for 72 h resulted in an increase in acetyl-CoA carboxylase activity to 166% of the controls. The simultaneous addition of 1 microgram/ml triiodothyronine and 10 mU/ml insulin caused the enzyme activity to rise to 240% of the controls. A dose-dependent reduction in acetyl-CoA carboxylase activity was brought about by 1 X 10(-4) to 1 X 10(-3) M dibutyryl cyclic AMP. The earliest effect of dibutyryl cyclic AMP was observed within 24 h. Glucose-6-phosphate dehydrogenase followed qualitatively the same pattern of response, whereas the constitutive enzyme, lactate dehydrogenase (EC 1.1.1.27), did not show significant changes in these experiments. The data demonstrate common features of hormonal regulation of lipogenesis in human fibroblasts with liver and adipose tissue and substantiate the growing evidence that thyroid hormones are of major importance for the regulation of this process.     

Reaction of cytochromes c and c2 with the Rhodobacter sphaeroides reaction center involves the heme crevice domain

In order to define the interaction domain on Rhodobacter sphaeroides cytochrome c2 for the photosynthetic reaction center, positively charged lysine amino groups on cytochrome c2 were modified to form negatively charged (carboxydinitrophenyl)- (CDNP-) lysines. The reaction mixture was separated into several different fractions by ion-exchange chromatography on (carboxymethyl)cellulose. Tryptic digests of these fractions were analyzed by reverse-phase peptide mapping to determine the lysines that had been modified. Fraction A was found to consist of a mixture of singly labeled derivatives modified at lysine-35, -88, -95, -97, and -105 and several other unidentified lysines comprising 32% of the total. Although it was not possible to resolve these derivatives, all of the identified lysines are located on the front surface of cytochrome c2 near the heme crevice. The second-order rate constant for the reaction of native cytochrome c2 with reaction centers was 2.0 X 10(8) M-1 s-1, while that for fraction A was 20-fold less, 1.0 X 10(7) M-1 s-1. This suggests that lysines surrounding the heme crevice of cytochrome c2 are involved in electrostatic interactions with carboxylate groups at the binding site of the reaction center. The reaction rates of horse heart cytochrome c derivatives modified at single lysine amino groups with trifluoroacetyl or trifluoromethylphenylcarbamoyl were also measured. Modification of lysine-8, -13, -27, -72, -79, and -87 surrounding the heme crevice significantly lowered the rate of reaction, while modification of lysines in other regions had no effect. This indicates that the reaction of horse heart cytochrome c with the reaction center also involves the heme crevice domain.     

The interaction of covalently bound heme with the cytochrome c maturation protein CcmE

The heme chaperone CcmE is a novel protein that binds heme covalently via a histidine residue as part of its essential function in the process of cytochrome c biogenesis in many bacteria as well as plant mitochondria. In the continued absence of a structure of the holoform of CcmE, identification of the heme ligands is an important step in understanding the molecular function of this protein and the role of covalent heme binding to CcmE during the maturation of c-type cytochromes. In this work, we present spectroscopic data that provide insight into the ligation of the heme iron in the soluble domain of CcmE from Escherichia coli. Resonance Raman spectra demonstrated that one of the heme axial ligands is a histidine residue and that the other is likely to be Tyr134. In addition, the properties of the heme resonances of the holo-protein as compared with those of a form of CcmE with non-covalently bound heme provide evidence for the modification of one of the heme vinyl side chains by the protein, most likely the 2-vinyl group.     

Calcium-dependent conformation of a heme and fingerprint peptide of the diheme cytochrome c peroxidase from Paracoccus pantotrophus

The structural changes in the heme macrocycle and substituents caused by binding of Ca(2+) to the diheme cytochrome c peroxidase from Paracoccus pantotrophus were clarified by resonance Raman spectroscopy of the inactive fully oxidized form of the enzyme. The changes in the macrocycle vibrational modes are consistent with a Ca(2+)-dependent increase in the out-of-plane distortion of the low-potential heme, the proposed peroxidatic heme. Most of the increase in out-of-plane distortion occurs when the high-affinity site I is occupied, but a small further increase in distortion occurs when site II is also occupied by Ca(2+) or Mg(2+). This increase in the heme distortion explains the red shift in the Soret absorption band that occurs upon Ca(2+) binding. Changes also occur in the low-frequency substituent modes of the heme, indicating that a structural change in the covalently attached fingerprint pentapeptide of the LP heme occurs upon Ca(2+) binding to site I. These structural changes may lead to loss of the sixth ligand at the peroxidatic heme in the semireduced form of the enzyme and activation.     

Intermediates generated during the reaction of reduced Rhodobacter sphaeroides cytochrome c oxidase with dioxygen

Cytochrome oxidase is one of the functionally most intriguing redox-driven proton pumps. During the last decade our increased understanding of the system has greatly benefited from theoretical calculations and modeling in the framework of three-dimensional structures of cytochrome c oxidases from different species. Because these studies are based on results from experiments, it is important that any ambiguities in the conclusions extracted from these experiments are discussed and elucidated. In a recent study Szundi et al. (Szundi et al. Biochemistry 2012, 51, 9302) investigated the reaction of the reduced Rhodobacter sphaeroides cytochrome c oxidase with O₂ and arrived at conclusions different from those derived from earlier investigations. In this short communication we compare these very recent data to those obtained from earlier studies and discuss the origin of the differences.     

Generation,characterization and crystallization of a cytochrome c1-subunit IV fused cytochrome bc1 complex from Rhodobacter sphaeroides

Cytochrome bc₁ complex catalyzes the reaction of electron transfer from ubiquinol to cytochrome c (or cytochrome c₂) and couples this reaction to proton translocation across the membrane. Crystallization of the Rhodobacter sphaeroides bc₁ complex resulted in crystals containing only three core subunits. To mitigate the problem of subunit IV being dissociated from the three-subunit core complex during crystallization, we recently engineered an R. sphaeroides mutant in which the N-terminus of subunit IV was fused to the C-terminus of cytochrome c₁ with a 14-glycine linker between the two fusing subunits, and a 6-histidine tag at the C-terminus of subunit IV (c₁-14Gly-IV-6His). The purified fusion mutant complex shows higher electron transfer activity, more structural stability, and less superoxide generation as compared to the wild-type enzyme. Preliminary crystallization attempts with this mutant complex yielded crystals containing four subunits and diffracting X-rays to 5.5 Å resolution.     

Mutants of the CuA site in cytochrome c oxidase of Rhodobacter sphaeroides: I. Spectral and functional properties

To study the functional significance of the unusual bimetallic Cu(A) center of cytochrome c oxidase, the direct ligands of the Cu(A) center in subunit II of the holoenzyme were mutated. Two of the mutant forms, M263L and H260N, exhibit major changes in activity (10% and 1% of wild-type, respectively) and in near-infrared and EPR spectra, but metal analysis shows that both mutants retain two coppers in the Cu(A) center and both retain proton pumping activity. In M263L, multifrequency EPR studies indicate the coppers are still electronically coupled, while all the other metal centers in M263L appear unchanged, by visible, EPR, and FTIR spectroscopy. Nevertheless, heme a3 is very slow to reduce with cytochrome c or dithionite under stopped-flow and steady-state conditions. This effect appears to be secondary to the change in redox equilibrium between Cu(A) and heme a. The studies reported here and in Wang et al. [Wang, K., Geren, L., Zhen, Y., Ma, L., Ferguson-Miller, S., Durham, B., and Millett, F. (2002) Biochemistry 41, 2298-2304] demonstrate that altering the ligands of Cu(A) can influence the rate and equilibrium of electron transfer between Cu(A) and heme a, but that the native ligation state is not essential for proton pumping.     

Physical interaction of CcmC with heme and the heme chaperone CcmE during cytochrome c maturation

Biogenesis of c-type cytochromes requires the covalent attachment of heme to the apoprotein. In Escherichia coli, this process involves eight membrane proteins encoded by the ccmABCDEFGH operon. CcmE binds heme covalently and transfers it to apocytochromes c in the presence of other Ccm proteins. CcmC is necessary and sufficient to incorporate heme into CcmE. Here, we report that the CcmC protein directly interacts with heme. We further show that CcmC co-immunoprecipitates with CcmE. CcmC contains two conserved histidines and a signature sequence, the so-called tryptophan-rich motif, which is the only element common to cytochrome c maturation proteins of bacteria, archae, plant mitochondria, and chloroplasts. We report that mutational changes of these motifs affecting the function of CcmC in cytochrome c maturation do not influence heme binding of CcmC. However, the mutants are defective in the CcmC-CcmE interaction, suggesting that these motifs are involved in the formation of a CcmC-CcmE complex. We propose that CcmC, CcmE, and heme interact directly with each other, establishing a periplasmic heme delivery pathway for cytochrome c maturation.