Crystal Structure of Oxidized Bacillus pasteurii Cytochrome c₅₅₃ at 0.97-Å Resolution

Abstract

This article reports the first X-ray structure of the soluble form of a c-type cytochrome isolated from a Gram-positive bacterium. Bacillus pasteurii cytochrome c(553), characterized by a low reduction potential and by a low sequence homology with cytochromes from Gram-negative bacteria or eukaryotes, is a useful case study for understanding the structure-function relationships for this class of electron-transfer proteins. Diffraction data on a single crystal of cytochrome c(553) were obtained using synchrotron radiation at 100 K. The structure was determined at 0.97-A resolution using ab initio phasing and independently at 1.70 A in an MAD experiment. In both experiments, the structure solution exploited the presence of a single Fe atom as anomalous scatterer in the protein. For the 0.97-A data, the phasing was based on a single data set. This is the most precise structure of a heme protein to date. The crystallized cytochrome c(553) contains only 71 of the 92 residues expected from the intact protein sequence, lacking the first 21 amino acids at the N-terminus. This feature is consistent with previous evidence that this tail, responsible for anchoring the protein to the cytoplasm membrane, is easily cleaved off during the purification procedure. The heme prosthetic group in B. pasteurii cytochrome c(553) is surrounded by three alpha-helices in a compact arrangement. The largely exposed c-type heme group features a His-Met axial coordination of the Fe(III) ion. The protein is characterized by a very asymmetric charge distribution, with the exposed heme edge located on a surface patch devoid of net charges. A structural search of a representative set of protein structures reveals that B. pasteurii cytochrome c(553) is most similar to Pseudomonas cytochromes c(551), followed by cytochromes c(6), Desulfovibrio cytochrome c(553), cytochromes c(552) from thermophiles, and cytochromes c from eukaryotes. Notwithstanding a low sequence homology, a structure-based alignment of these cytochromes shows conservation of three helical regions, with different additional secondary structure motifs characterizing each protein. In B. pasteurii cytochrome c(553), these motifs are represented by the shortest interhelix connecting fragments observed for this group of proteins. The possible relationships between heme solvent accessibility and the electrochemical reduction potential are discussed.

Theoretical study of structure of catalytic copper site in nitrite reductase

“…The results showed that we may improve the structure locally in terms of the R free factor. Moreover, we have shown [53] that refinement with ComQum-X of a medium-resolution (170 pm) crystal structure of cytochrome c 553 brings the geometry of the heme group and its ligands closer to that observed in an atomic-resolution structure (97 pm) of the same protein [54]. For example, the errors in the Fe-ligand distances are reduced from 3-9, 12, and 32 pm to 1, 0, and 2 pm (for the porphyrin, His, and methionine ligands, respectively).…”

Structural Characterization of Nitrosomonas europaea Cytochrome c‐552 Variants with Marked Differences in Electronic Structure

“…The heme groups are well ordered, and the structures well refined, as demonstrated by limited negative and positive electron density in the (F o ÀF c ) difference maps shown for the heme regions in Figure 3. The location and identity of iron within the heme group is corroborated by a representative 14 s anomalous difference Fourier map from the Ne c-552r structure (purple mesh in Figure 3 A) in which the signal exceeded 26 s. The overall structures are PDB (resolution) [c] B. pasteureii c-553 3.36 [18] 0.35 1C75 (0.97 ) [19] Ne c-552r 3.34 [15] 0.62 4JCG (1.63 ) Ht c-552 3.23 [20] 0.62 1YNR (2.00 ) [21] Pa c-551 3.20 [15] 0.49 351C (1.60 ) [22] Ne N64D 3.13 [15] 0.71 [a] 3ZOX (2.1 ) Horse cyt c 3.06 [15] 1.00 1HRC (1.90 ) [5] [a] Average of values for the four monomers for the monoclinic crystal form. .…”

Low‐Temperature EPR and Mössbauer Spectroscopy of Two Cytochromes with His–Met Axial Coordination Exhibiting HALS Signals

“…Like many other cytochromes c, N. europaea c 552 and B. pasteurii c 553 have histidine (His) and methionine (Met) as ligands of the heme Fe, [31,35] as shown in Figure 1. [36,37] The 0.95 X-ray resolution of cytochrome c 553 provides the most accurate model so far available for a c-type cytochrome. [37] Cytochrome c 552 is a small (9.1 kDa) soluble protein [28,36,38,39] involved in several physiological reactions, including the reduction of the diheme cytochrome peroxidase [28] and possibly the terminal cytochrome oxidase.…”

“…[36,37] The 0.95 X-ray resolution of cytochrome c 553 provides the most accurate model so far available for a c-type cytochrome. [37] Cytochrome c 552 is a small (9.1 kDa) soluble protein [28,36,38,39] involved in several physiological reactions, including the reduction of the diheme cytochrome peroxidase [28] and possibly the terminal cytochrome oxidase. [38] In vitro cytochrome c 552 is able to oxidize the tetraheme cytochrome c 554 , the electron acceptor of the 24-heme hydroxylamine oxidase (HAO), [31,32,[38][39][40] while cytochrome c 553 (molecular weight 9.6 kDa) [33,34] is proposed to be part of the electron flow chain to a terminal oxidase.…”

“…[36] Such a low potential is not due to enthalpic effects related to electronic, covalent, or electrostatic parameters, but rather derives from a very large negative change in entropy during reduction, ascribed to a rearrangement of water molecules in the vicinity of the largely solvent-exposed heme. [35,37] Even though the physiological function of cytochrome c 552 is still unclear, the low reduction potential, which is common for most c-type cytochromes from alkaliphilic bacteria, [41,42] may facilitate the electron transfer to the terminal oxidase in the presence of a large negative membrane potential due to the alkalinity of the growth medium. [43] Another peculiar difference between the ferric forms of cytochrome c 552 and cytochrome c 553 involves their paramagnetic NMR spectra.…”