Crystallization and preliminary structure determination of the membrane-bound complex cytochromecnitrite reductase fromDesulfovibrio vulgarisHildenborough

Abstract

The cytochrome c nitrite reductase (cNiR) isolated from Desulfovibrio vulgaris Hildenborough is a membrane-bound complex formed of NrfA and NrfH subunits. The catalytic subunit NrfA is a soluble pentahaem cytochrome c that forms a physiological dimer of about 120 kDa. The electron-donor subunit NrfH is a membrane-anchored tetrahaem cytochrome c of about 18 kDa molecular weight and belongs to the NapC/NirT family of quinol dehydrogenases, for which no structures are known. Crystals of the native cNiR membrane complex, solubilized with dodecylmaltoside detergent (DDM), were obtained using PEG 4K as precipitant. Anomalous diffraction data were measured at the Swiss Light Source to 2.3 Å resolution. Crystals belong to the orthorhombic space group P2 1 2 1 2 1 , with unit-cell parameters a = 79.5, b = 256.7, c = 578.2 Å . Molecularreplacement and MAD methods were combined to solve the structure. The data presented reveal that D. vulgaris cNiR contains one NrfH subunit per NrfA dimer.

A needle in a haystack: The active site of the membrane‐bound complex cytochrome c nitrite reductase

“…The proteins from Desulfovibrio desulfuricans and Desulfovibrio vulgaris Hildenborough are co-purified with their physiological electron donor, the tetraheme NrfH, as a 2:1 complex (NrfA 2 NrfH) which contains 14 hemes [5] and dissociate under denaturing conditions [5,6]. The very recently determined structure of the complex from D. vulgaris Hildenborough confirms the overall stoichiometry and suggests a physiological NrfA 4 NrfH 2 organization [7,8].…”

Reductive activation of the heme iron–nitrosyl intermediate in the reaction mechanism of cytochrome c nitrite reductase: a theoretical study

“…High turnover rates for nitrite reduction by CcNiR (415-962 s -1 [3]) suggest very efficient proton and electron transfer processes that must be guided and supported by the protein. The X-ray crystal structures of CcNiR from Escherichia coli [4], Wolinella succinogenes [5], Sulfurospirillum deleyianum [6], Desulfovibrio desulfuricans [7,8], Desulfovibrio vulgaris [9][10][11], and Thialkalivibrio nitratireducens [12] have been solved. All crystallographically characterized enzymes, except that of T. nitratireducens (TvNiR), exist as homodimers, where each monomer contains five covalently attached c-type hemes and two calcium atoms.…”

Quinol Oxidation by c-Type Cytochromes: Structural Characterization of the Menaquinol Binding Site of NrfHA

“…1). The HQNO-soaked crystal has smaller unit cell dimensions compared with the native crystals, 19 and only one NrfH 2 A 4 biological unit is present in the crystallographic asymmetric unit (instead of three as in the native crystal). The present crystallographic model was refined to final R and R free values of 22.0% and 26.1%, respectively.…”

“…9,13 The resolution of the ligand-bound structures may also be significantly lower than that of the free complexes. 11 A large needle of ∼ 1.2 × 0.08 × 0.06 mm 3 appeared in 9% PEG 4K, 0.1 M Hepes, pH 7.5, and 100 mM glycyl-glycyl- 19 The relatively high crystal mosaic spread (0.7°-0.8°), which had to be fixed in data processing due to the presence of split spots, limited the overall completeness of data to 86.5% (75.4% in the highest-resolution shell). Data were processed and merged with HKL2000.…”

“…19 In summary, crystals were grown at 277 K in 9%-12% polyethylene glycol (PEG) 4K, 0.1 M Hepes, pH 7.5, and 30-100 mM glycyl-glycyl-glycine, using the vapor diffusion method.…”