Characterization of Rhodobacter sphaeroides Cytochrome c2 Proteins with Altered Heme Attachment Sites

Rı́os-Velázquez, Carlos; Cox, Rebecca L.; Donohue, Timothy J.

doi:10.1006/abbi.2001.2330

Cited by 17 publications

(17 citation statements)

References 35 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The crucial difference between our failure to observe covalent attachment of heme to apocytochromes with a CXXAH or AXXCH heme-binding motif and previous related studies (20,21) is that in the earlier cases the products were unstable with respect to degradation. Thus is was not possible to determine unequivocally whether heme was in fact covalently attached by the bacterial Ccm system before the protein degraded.…”

Section: Discussioncontrasting

confidence: 84%

“…A further interpretation, which may or may not be combined with the obligate formation of one or more disulfide bonds, is that the ultimate highly specific recognition determinant for heme attachment by the Ccm system is the two cysteine thiols in the apocytochrome heme-binding motif. Note, however, that any rationalization of our data must allow for the fact that the Ccm proteins are active with substrate apocytochromes that either have naturally (43), or have as the result of amino acid substitutions (20), CXXXXCH or CXXXCH heme binding-motifs, and thus it is the two cysteines that are important rather than their precise spatial arrangement.…”

Section: Discussionmentioning

confidence: 99%

“…Among other things, this would provide strong evidence as to whether an intramolecular disulfide bond in an apocytochrome c is indeed an intermediate. Ríos-Velá zquez et al (20) made single and double cysteine to alanine substitutions in the heme-binding motif of Rhodobacter sphaeroides cytochrome c 2 . Such variant proteins were unable to support growth via photosynthesis where functional cytochrome c 2 is required.…”

mentioning

confidence: 99%

See 2 more Smart Citations

The Escherichia coli Cytochrome cMaturation (Ccm) System Does Not Detectably Attach Heme to Single Cysteine Variants of an Apocytochrome c

Allen¹,

Tomlinson

Lin

et al. 2002

Journal of Biological Chemistry

View full text Add to dashboard Cite

Cytochromes c are typically characterized by the covalent attachment of heme to polypeptide through two thioether bonds with the cysteine residues of a Cys-XaaXaa-Cys-His peptide motif. In many Gram-negative bacteria, the heme is attached to the polypeptide by the periplasmically functioning cytochrome c maturation (Ccm) proteins. Exceptionally, Hydrogenobacter thermophilus cytochrome c 552 , which has a normal CXXCH heme-binding motif, and variants with AXXCH, CXXAH, and AXXAH motifs, can be expressed as stable holocytochromes in the cytoplasm of Escherichia coli. By targeting these proteins to the periplasm using a signal peptide, with or without co-expression of the Ccm proteins, we have assessed the ability of the Ccm system to attach heme to proteins with no, one, or two cysteine residues in the heme-binding motif. Only the wild-type protein, with two cysteines, was effectively processed and thus accumulated in the periplasm as a holocytochrome. This is strong evidence for disulfide bond formation involving the two cysteine residues of apocytochrome c as an intermediate in Ccm-type Gramnegative bacterial cytochrome c biogenesis and/or that only a pair of cysteines can be recognized by the heme attachment apparatus.

show abstract

Section: Discussioncontrasting

confidence: 84%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

See 1 more Smart Citation

The Escherichia coli Cytochrome cMaturation (Ccm) System Does Not Detectably Attach Heme to Single Cysteine Variants of an Apocytochrome c

Allen¹,

Tomlinson

Lin

et al. 2002

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…The apocytochrome c recognition motif for system I has been much studied and appears to be only the CXXCH motif (6-8, 13, 14, 29), with the caveat that CXXXCH may also be used (130). Some variants of system I (e.g., NrfEFG [51], which is related to CcmFGH) and system II (CcsBA) recognize slightly altered apocytochrome c signature motifs such as CXXCK (51,74,117) or CX15CH (73).…”

Section: System IV and Othersmentioning

confidence: 99%

CytochromecBiogenesis: Mechanisms for Covalent Modifications and Trafficking of Heme and for Heme-Iron Redox Control

Kranz¹,

Richard-Fogal²,

Taylor

et al. 2009

Microbiol Mol Biol Rev

235

372

View full text Add to dashboard Cite

SUMMARY Heme is the prosthetic group for cytochromes, which are directly involved in oxidation/reduction reactions inside and outside the cell. Many cytochromes contain heme with covalent additions at one or both vinyl groups. These include farnesylation at one vinyl in hemes o and a and thioether linkages to each vinyl in cytochrome c (at CXXCH of the protein). Here we review the mechanisms for these covalent attachments, with emphasis on the three unique cytochrome c assembly pathways called systems I, II, and III. All proteins in system I (called Ccm proteins) and system II (Ccs proteins) are integral membrane proteins. Recent biochemical analyses suggest mechanisms for heme channeling to the outside, heme-iron redox control, and attachment to the CXXCH. For system II, the CcsB and CcsA proteins form a cytochrome c synthetase complex which specifically channels heme to an external heme binding domain; in this conserved tryptophan-rich “WWD domain” (in CcsA), the heme is maintained in the reduced state by two external histidines and then ligated to the CXXCH motif. In system I, a two-step process is described. Step 1 is the CcmABCD-mediated synthesis and release of oxidized holoCcmE (heme in the Fe+3 state). We describe how external histidines in CcmC are involved in heme attachment to CcmE, and the chemical mechanism to form oxidized holoCcmE is discussed. Step 2 includes the CcmFH-mediated reduction (to Fe+2) of holoCcmE and ligation of the heme to CXXCH. The evolutionary and ecological advantages for each system are discussed with respect to iron limitation and oxidizing environments.

show abstract

“…Soluble and membrane fractions were obtained from cell lysates prepared by sonication of mid-exponential-phase aerobic cultures of R. sphaeroides (ϳ5 ϫ 10 8 cells/ml) (45). Periplasmic, cytoplasmic, inner, and outer membrane fractions were prepared by osmotic shock (53), using compartmentspecific enzymes to estimate purity (1,4,42).…”

mentioning

confidence: 99%

Features of Rhodobacter sphaeroides CcmFH

2003

View full text Add to dashboard Cite

In this study, the in vivo function and properties of two cytochrome c maturation proteins, CcmF and CcmH from Rhodobacter sphaeroides, were analyzed. Strains lacking CcmH or both CcmF and CcmH are unable to grow under anaerobic conditions where c-type cytochromes are required, demonstrating their critical role in the assembly of these electron carriers. Consistent with this observation, strains lacking both CcmF and CcmH are deficient in c-type cytochromes when assayed under permissive growth conditions. In contrast, under permissive growth conditions, strains lacking only CcmH contain several soluble and membrane-bound c-type cytochromes, albeit at reduced levels, suggesting that this bacterium has a CcmH-independent route for their maturation. In addition, the function of CcmH that is needed to support anaerobic growth can be replaced by adding cysteine or cystine to growth media. The ability of exogenous thiol compounds to replace CcmH provides the first physiological evidence for a role of this protein in thiol chemistry during c-type cytochrome maturation. The properties of R. sphaeroides cells containing translational fusions between CcmF and CcmH and either Escherichia coli alkaline phosphatase or ␤-galactosidase suggest that they are each integral cytoplasmic membrane proteins with their presumed catalytic domains facing the periplasm. Analysis of CcmH shows that it is synthesized as a higher-molecular-weight precursor protein with an N-terminal signal sequence.

show abstract

Characterization of Rhodobacter sphaeroides Cytochrome c2 Proteins with Altered Heme Attachment Sites

Cited by 17 publications

References 35 publications

The Escherichia coli Cytochrome cMaturation (Ccm) System Does Not Detectably Attach Heme to Single Cysteine Variants of an Apocytochrome c

The Escherichia coli Cytochrome cMaturation (Ccm) System Does Not Detectably Attach Heme to Single Cysteine Variants of an Apocytochrome c

CytochromecBiogenesis: Mechanisms for Covalent Modifications and Trafficking of Heme and for Heme-Iron Redox Control

Features of Rhodobacter sphaeroides CcmFH

Contact Info

Product

Resources

About