Ligand Binding and Covalent Structure of an Oxygen-Binding Heme Protein from Rhodobacter sphaeroides, a Representative of a New Structural Family of c-Type Cytochromes

Abstract

The amino acid sequence of an oxygen-binding heme protein (SHP) from Rhodobacter sphaeroides has been determined. The cysteines, which bind the single heme group in the 112-residue protein, are located at positions 43 and 46. SHP is similar in size to the large membrane-bound form of the class I cytochrome c5 of Azotobacter vinelandii (116 residues) and in the location of the heme binding site at positions 48 and 51. Two extra cysteines in SHP (residues 89 and 97) are located in positions similar to those of cytochrome c5 (residues 98 and 101) and form a disulfide bridge in both proteins. In total, four regions of alpha-helix are predicted, covering 46% of the protein, which is comparable to that in other small cytochromes. SHP is thus distantly related to small class I c-type cytochromes but is representative of a distinct family by virtue of its high-spin nature, the lack of a strong sixth ligand, and its capacity to bind oxygen. Potentially, the most important characteristic of SHP is its ability to transiently bind oxygen during autoxidation, which occurs with a half-life of 3 min with a 4-fold excess of O2. SHP also binds carbon monoxide, azide, and cyanide. The kinetics of reduction by free flavins indicate that SHP is less reactive than other class I cytochromes c and that the heme is less accessible to solvent. There is localized positive charge (+3) at the site of reduction of SHP, although the overall protein charge is -2. This may account in part for the ability of SHP to bind anions.

Crystal Structures of an Oxygen-binding Cytochrome cfrom Rhodobacter sphaeroides

“…Examples of class I cytochromes c include mitochondrial cytochrome c (23), the two domains of the Pseudomonas stutzeri diheme cytochrome c 4 (24), and the Azotobacter vinelandii cytochrome c 5 (25). Comparison of SHP to these cytochromes, by superimposing the heme groups, clearly indicates how the first ␣-helix in SHP (Ser 4 - Gly 17 ) is an extra N-terminal extension to the class I fold as was previously suggested (12). This helix is packed against the C-terminal or fourth helix (Gly , LeuVal-Phe 94 , and Tyr-Trp-Phe 97 (mitochondrial cytochrome c numbering)) are mainly responsible for the interaction between the two helices.…”

“…Unlike the cytochromes cЈ, SHP binds molecular oxygen as demonstrated by the absence of isosbestic points and the formation of a new transient spectral species during autooxidation. The half-life of the SHP-oxygen complex was determined to be 3 min (12), which is a dramatic difference to the observed half-life of approximately 1 day for the myoglobinoxygen complex (13). SHP is substantially less reactive with exogenous ligands than myoglobin.…”

“…This site has been implicated as the interaction site for redox proteins carrying a complementary negative charge (32). As was already demonstrated (12), SHP carries a positive charge of approximately ϩ3 at the site of reduction. The conserved residues Lys 61 , Arg 80 , and Arg 87 are clearly responsible for this concentrated positive charge at the exposed heme edge.…”

“…In SHP, this entire region is folded in one slightly bent ␣-helix (Asp 77 -Ile 93 ) oriented parallel to the heme plane. The charged Glu 82 side chain forms a strong hydrogen bond to Tyr 12 and a salt bridge with Lys 102 , thus effectively tying the beginning of this helix to both the first and the last ␣-helix. These residues are conserved in the known SHP-like sequences, which underlies the importance of this structural feature.…”

“…The protein is not homologous to any of the above mentioned gas-binding heme proteins. The amino acid sequence actually indicates a distant relationship to the class I cytochromes c (12). SHP has a high spin heme, indicating lack of a strong field sixth ligand, which confers to SHP spectral properties similar to those of the globins and of cytochromes cЈ.…”