³¹P Nuclear magnetic resonance study of the flavoprotein component of the Escherichia coli sulfite reductase

Abstract

SiR-FP60, the monomeric form of the Escherichia coli sulfite reductase flavoprotein component (SiR-FP), has been analysed by 31 P-NMR spectroscopy. This protein was reported previously as a reliable simplified model for native SiR-FP [Zeghouf, M., Fontecave, M., Macherel, D., & Cove Ás, J. (1998) Biochemistry 37, 6117±6123]. SiR-FP60 was examined in its native form, as a complex with NADP + and after monoelectronic reduction either with NADPH or dithionite. In these latter cases, the stabilized FMN semiquinone radical offers a natural and internal paramagnetic probe. The paramagnetic effect of added manganese was also studied. In each case, the NMR parameters were extracted from digitalized data by a deconvolution procedure and compared with those obtained previously with cytochrome P450 reductase. Evolution of the NMR parameters and of calculated relaxation rate constants upon biochemical modifications of SiR-FP60 led us to propose that the reactive center is more compact than the one of cytochrome P450 reductase, with the redox components, FMN, FAD and NADPH, in a tighter spatial arrangement, close to the protein surface. This underlies some subtle differences between the two proteins for which a very similar overall structure is likely considering their common genetic origin and common operating cycle.Keywords: sulfite reductase; cytochrome P450 reductase; 31 P-NMR; protein structure; Escherichia coli.Escherichia coli sulfite reductase (SiR) is a multimeric and soluble hemoflavoprotein with a molecular mass of 780 kDa and an a 8 b 4 quaternary structure. Subunit a is the flavoprotein component (SiR-FP) of the enzyme. It contains one NADPHbinding domain and two distinct flavinic domains: an N-terminal flavodoxin-like domain that binds one FMN and a C-terminal ferredoxin-NADP + reductase-like, FAD-containing domain [1,2]. This was strongly suggested from sequence comparisons, limited proteolysis experiments and expression of the isolated FMNbinding domain [1±3]. During the physiological turnover leading to reduction of sulfite, the first event is an hydride transfer from NADPH to bound FAD. Electrons are then transferred to FMN before being transferred one at a time to the b-subunits, which form the hemoprotein component (SiR-HP), where the six-electron reduction of sulfite to sulfide takes place [4,5].These characteristics, i.e. a bi-flavin domain, NADPH as the electron donor and, a metal center as the electron acceptor, are shared by all the members of the SiR-FP family that includes mammalian NADPH-cytochrome P450 reductases, nitric oxide synthase (NOS) and Bacillus megaterium cytochrome P450 (P450BM-3) [6,7]. Among these proteins, only the structure of cytochrome P450 reductase is known [8] but amino-acid sequence analysis and structural alignment with flavodoxin and ferredoxin reductase predict an overall structure very similar to that of cytochrome P450 reductase for all the members of the family [8]. The hypothesis that SiR-FP belongs to this family of proteins was strengthened by the recent demonstrati...

Phosphorylation and Flexibility of Cyclic-AMP-Dependent Protein Kinase (PKA) Using 31P NMR Spectroscopy

“…Sample Preparation. cAMP-dependent protein kinase Cα catalytic subunit was solubly expressed in E. coli and purified using a PKI( − ) affinity column as previously described (). The different phosphorylated forms were separated by ion exchange chromatography (Mono-S, Pharmacia) using a LiCl gradient from 0 to 300 mM LiCl in 20mM bis-tris propane at pH 8.5.…”

“…31 P NMR spectroscopy (4) is thus ideally suited for studies of phosphorylated or phosphate binding molecules, and allows the identification of specific phosphorylation sites, kinetics of phosphotransfer events, or analysis of dynamic phenomena involving phosphorus, on time scales ranging from picoseconds to days. Examples include studies of amino acids (5), peptides (6), alkaline phosphatase (7), the complex of G-actin with ATP (8,9), flavoprotein component of the Escherichia coli sulfite reductase (10), bovine cardiac troponin T and I (11,12), and other proteins (13)(14)(15)(16)(17). Several studies have focused on the application of 31 P NMR to evaluate internal flexibilities, for example of DNA (18) and also of proteins (17,19).…”

Structure–Function Relationships in the Oligomeric NADPH-Dependent Assimilatory Sulfite Reductase

“…SiRFP-60 is a monomeric form of Fld1 and FNR1, without the octamerization domain. (C) Model of SiRFP based on a nuclear magnetic resonance structure of Fld1 and an X-ray crystal structure of FNR1, superimposed on the closed conformation of CPR . The domains are colored as in panel A, but neither the N-terminus, which is responsible for SiRFP octamerization, nor the flexible linker joining the domains is pictured.…”

Evolution of bioinorganic motifs in P450-containing systems

“…?, [ l l ] ; the recent experiments, however, strongly suggest the a$, structure [31]. The notable structural differences of SIR-FP include a more compact spatial arrangement of FMN, FAD and NADPH than in CPR [32] and disordered Fld domain [33]. The purified SIR-FP can act as CPR in nitro, supporting the CYP17-catalysed 17ahydroxylation of pregnenolone [34].…”