NMR Solution Structure, Backbone Mobility, and Homology Modeling ofc-Type Cytochromes from Gram-Positive Bacteria

Abstract

The solution structure of oxidized cytochrome c(553) (71 amino acid residues) from the Gram-positive bacterium Bacillus pasteurii is here reported and compared with the available crystal structure. The solution structure is obtained from 1609 meaningful NOE data (22.7 per residue), 76 dihedral angles, and 59 pseudocontact shifts. The root mean square deviations from the average structure are 0.25+/-0.07 and 0.59+/-0.13 A for the backbone and all heavy atoms, respectively, and the quality assessment of the structure is satisfactory. The solution structure closely reproduces the fold observed in the crystal structure. The backbone mobility was then investigated through amide (15)N relaxation rate and (15)N-(1)H NOE measurements. The protein is rigid in both the sub-nanosecond and millisecond time scales, probably due to the relatively large heme:number of amino acids ratio. Modeling of eight c-type cytochromes from other Gram-positive bacteria with a high sequence identity (>30 %) to the present cytochrome c(553) was performed. Analysis of consensus features accounts for the relatively low reduction potential as being due to extensive heme hydration and indicates residues 34-35, 44-46, 69-72, and 75 as a conserved hydrophobic patch for the interaction with a protein partner. At variance with mitochondrial c-type cytochrome, this protein does not experience pH-dependent coordination equilibria. The reasons for this difference are analyzed.

Solution Structure of a Monoheme Ferrocytochrome c from Shewanella putrefaciens and Structural Analysis of Sequence-Similar Proteins: Functional Implications

“…On the other hand, the presently proposed interaction patch is essentially hydrophobic. A similar contention has been proposed for other bacterial cytochromes, based on their structural features ( − ). It is also relevant to observe that NMR studies of the interaction between plastocyanin and cytochrome f point out that hydrophobic interactions play an important role in determining the correct orientation of the complex ( , ).…”

Solution Structure of a Monoheme Ferrocytochrome c from Shewanella putrefaciens and Structural Analysis of Sequence-Similar Proteins: Functional Implications

“…On the other hand, the presently proposed interaction patch is essentially hydrophobic. A similar contention has been proposed for other bacterial cytochromes, based on their structural features ( − ). It is also relevant to observe that NMR studies of the interaction between plastocyanin and cytochrome f point out that hydrophobic interactions play an important role in determining the correct orientation of the complex ( , ).…”

Comparison of the backbone dynamics of wild-type Hydrogenobacter thermophilus cytochrome c 552 and its b-type variant

“…The lowest order parameters are seen in the loop region between helices 1 and 2 and at the C-terminus. Similar results have been reported for a number of other proteins adopting the α-helical fold of class I c -type cytochromes, especially when the iron is in its reduced state (Banci et al 2002 ; Barker et al 2001 ; Bartalesi et al 2003 ; Cordier et al 1998 ; Fetrow and Baxter 1999 ; Liu et al 2009 ; Russell et al 2003 ; Ubbink et al 1996 ). In general, any significant fast timescale backbone motions are restricted to exposed residues in loop regions and the protein termini, with the four helices forming a rigid core.…”

The stability of the cytochrome c scaffold as revealed by NMR spectroscopy

“…In the absence of detectable NOEs, 15 N-1 H HSQC experiments are the best tool for acquiring information on residual protein structure. Upon increasing GdmCl concentration the peaks of the native protein in HSQC experiments decrease in intensity, while those of a new species increase.…”

Solution Structure of a Monoheme Ferrocytochrome c from Shewanella putrefaciens and Structural Analysis of Sequence-Similar Proteins: Functional Implications

“…On the other hand, the presently proposed interaction patch is essentially hydrophobic. A similar contention has been proposed for other bacterial cytochromes, based on their structural features ( − ). It is also relevant to observe that NMR studies of the interaction between plastocyanin and cytochrome f point out that hydrophobic interactions play an important role in determining the correct orientation of the complex ( , ).…”

Comparison of the backbone dynamics of wild-type Hydrogenobacter thermophilus cytochrome c 552 and its b-type variant

“…The lowest order parameters are seen in the loop region between helices 1 and 2 and at the C-terminus. Similar results have been reported for a number of other proteins adopting the α-helical fold of class I c -type cytochromes, especially when the iron is in its reduced state (Banci et al 2002 ; Barker et al 2001 ; Bartalesi et al 2003 ; Cordier et al 1998 ; Fetrow and Baxter 1999 ; Liu et al 2009 ; Russell et al 2003 ; Ubbink et al 1996 ). In general, any significant fast timescale backbone motions are restricted to exposed residues in loop regions and the protein termini, with the four helices forming a rigid core.…”

The stability of the cytochrome c scaffold as revealed by NMR spectroscopy

“…In the absence of detectable NOEs, 15 N-1 H HSQC experiments are the best tool for acquiring information on residual protein structure. Upon increasing GdmCl concentration the peaks of the native protein in HSQC experiments decrease in intensity, while those of a new species increase.…”

Solution Structure of a Monoheme Ferrocytochrome c from Shewanella putrefaciens and Structural Analysis of Sequence-Similar Proteins: Functional Implications

“…On the other hand, the presently proposed interaction patch is essentially hydrophobic. A similar contention has been proposed for other bacterial cytochromes, based on their structural features ( − ). It is also relevant to observe that NMR studies of the interaction between plastocyanin and cytochrome f point out that hydrophobic interactions play an important role in determining the correct orientation of the complex ( , ).…”

Comparison of the backbone dynamics of wild-type Hydrogenobacter thermophilus cytochrome c 552 and its b-type variant

“…The lowest order parameters are seen in the loop region between helices 1 and 2 and at the C-terminus. Similar results have been reported for a number of other proteins adopting the α-helical fold of class I c -type cytochromes, especially when the iron is in its reduced state (Banci et al 2002 ; Barker et al 2001 ; Bartalesi et al 2003 ; Cordier et al 1998 ; Fetrow and Baxter 1999 ; Liu et al 2009 ; Russell et al 2003 ; Ubbink et al 1996 ). In general, any significant fast timescale backbone motions are restricted to exposed residues in loop regions and the protein termini, with the four helices forming a rigid core.…”

The stability of the cytochrome c scaffold as revealed by NMR spectroscopy

“…In the absence of detectable NOEs, 15 N-1 H HSQC experiments are the best tool for acquiring information on residual protein structure. Upon increasing GdmCl concentration the peaks of the native protein in HSQC experiments decrease in intensity, while those of a new species increase.…”