Cytochrome cd 1 Structure: unusual haem environments in a nitrite reductase and analysis of factors contributing to β-propeller folds 1 1Edited by K. Nagai

Demonstration of Proton-coupled Electron Transfer in the Copper-containing Nitrite Reductases

“…xylosoxidans (NCIMB 11015, AxNiR) was overexpressed in Escherichia coli BL21(DE3) cells, and the purification was similar to the method described previously (42,43) (see supplemental material for detailed description). AxNiR concentrations were determined using an extinction coefficient of 4 Very recently spectroscopic and computational methods have shown that ET from the T1 to the T2Cu sites in the green copper enzyme RsNiR (Rhodobacter sphaeroides) requires the initial proton transfer from the Asp residue in the active site pocket to nitrite thereby triggering the reductive cleavage of the substrate (40,41 ⑀ 595 ϭ 6.3 mM Ϫ1 cm Ϫ1 (9,18). The AxNiR enzyme lacking the copper atom in the T2Cu center (T2DNiR) was expressed and purified in the same way as AxNiR except that the dialysis steps into CuSO 4 were omitted (27,42,44).…”

“…With reported onrates for NO binding to deoxyHb of ϳ2. 4 (50), this indicator reaction is expected to be a useful reporter for the AxNiR turnover reaction. Moreover, the reaction rate of nitrite with heme proteins has been reported to be 10,000 times smaller than that of NO (51).…”

“…The relevance of these active site residues, however, as well as the timing of the two protonation steps is still a matter of debate (35,40,41). 4 There are no experimental studies that have been aimed at directly examining the kinetic coupling of PT and ET steps in AxNiR. In this study of the blue AxNiR, our aims were to gain further insight into the mechanism of nitrite reduction by combining multiple turnover experiments with laser photoexcitation studies to measure the (single turnover) inter-copper ET.…”

Cell surface proteins in archaeal and bacterial genomes comprising “LVIVD”, “RIVW” and “LGxL” tandem sequence repeats are predicted to fold as β-propeller

“…The most common "Velcro" system is composed of first three inner strands from the C-terminal end of the polypeptide chain and the fourth outer strand from the N-terminus (1 + 3 arrangement) as in G protein subunit (PDB ID: 1GOT) [15]. The other combinations could either be a 3 + 1 arrangement as in cytochrome cd1 nitrite reductase (PDB ID: 1QKS) [16,17], or a 2 + 2 arrangement as in regulator of chromosome condensation (PDB ID: 1A12) [18]. The lack of molecular velcro in the 7 bladed non-catalytic domain of prolyl oligopeptidase permits partial opening of the ␤-propeller domain which regulates the proteolysis of peptides (PDB ID: 1QFM) [19].…”

The automatic detection of known β-propeller structural motifs from protein tertiary structure

“…The ␤-propellers are classified into five different types according to the number of 'blades' (or ␤-sheets) that are arranged radially about a pseudosymmetry axis and a sixth type classified according to the secondary structure composition of the blades (the ␤␤␣␤ molecular unit). The ␤-propeller fold is stabilized either through molecular 'Velcro', in which ring closure is achieved by forming anti-parallel ␤-strands between the termini of the propeller domain as in cytochrome cd1 nitrite reductase [11,12] or through predominant hydrophobic interactions between the ␤-sheets, or through a disulfide bond between the first and last blades as in hemopexin [13,14]. A model for multi-sheet packing in the ␤-propeller fold proposed earlier gives geometrical parameters of the ␤-propellers composed of different numbers of sheets and patterns of residue packing at their sheet-to-sheet interfaces [15].…”