Recruitment of governing elements for electron transfer in the nitric oxide synthase family

Abstract

At least three building blocks are responsible for the molecular basis of the modulation of electron transfer in nitric oxide synthase (NOS) isoforms: the calmodulin-binding sequence, the C-terminal extension, and the autoregulatory loop in the reductase domain. We have attempted to impart the control conferred by the C termini of NOS to cytochrome P450 oxidoreductase (CYPOR), which contains none of these regulatory elements. The effect of these C termini on the properties of CYPOR sheds light on the possible evolutionary origin of NOS and addresses the recruitment of new peptides on the development of new functions for CYPOR. The C termini of NOSs modulate flavoprotein-mediated electron transfer to various electron acceptors. The reduction of the artificial electron acceptors cytochrome c, 2,6-dichlorophenolindophenol, and ferricyanide was inhibited by the addition of any of these C termini to CYPOR, whereas the reduction of molecular O(2) was increased. This suggests a shift in the rate-limiting step, indicating that the NOS C termini interrupt electron flux between flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) and/or the electron acceptors. The modulation of CYPOR by the addition of the NOS C termini is also supported by flavin reoxidation and fluorescence-quenching studies and antibody recognition of the C-terminal extension. These experiments support the origin of the NOS enzymes from modules consisting of a heme domain and CYPOR or ferredoxin-NADP(+) reductase- and flavodoxin-like subdomains that constitute CYPOR, followed by further recruitment of smaller modulating elements into the flavin-binding domains.

Fig. 1.

Ribbon diagrams of flavodoxin (light pink), ferredoxin-NADP+ reductase (FNR; dark pink), CYPOR (green), and the reductase domain of rat nNOS (dark purple). (A) Overlay of flavodoxin and FNR onto the CYPOR structure. (B) Overlay of CYPOR and the reductase domain of nNOS. The three major structural differences between nNOS and CYPOR are shown: the C-terminal extension of nNOS (CT), the autoregulatory insert (AI), and β-finger (BF). Dotted lines indicate segments that are not visible in the crystal structure of the reductase domain of nNOS.

Fig. 2.

Alignment of C-terminal amino acid sequences of rat CYPOR, bovine eNOS, rat nNOS and murine iNOS.

Fig. 3.

Detection of eNOS C terminus by a monoclonal antibody, directed to the intact enzyme tail. Enzyme preparations (lanes a–f) were subjected to 10% SDS/PAGE and either Coomassie-stained (A) or subjected to Western analysis (B), where immunoreactive protein was visualized via alkaline phosphatase reagent. Lanes: a and b, full-length CYPOR; c, CYPOR-iNOS^T; d, CYPOR-nNOS^T; e, CYPOR-eNOS^T; f, eNOS, which was partially degraded so that sensitivity of the antibody to protein fragments could be assessed. Only CYPOR-eNosT reacted with the monoclonal antibody.

Fig. 4.

Fluorescence emission spectra of CYPOR, and CYPOR with nNOS, eNOS, and iNOS tails. The relative emission intensities at the λ_max of the spectra were 185,280, 52,140, 33,130, and 30,070 counts per second for CYPOR, CYPOR-iNOS^T, CYPOR-eNOS^T, and CYPOR-nNOS^T tails, respectively. The λ_max values for the maxima were 530 nm (CYPOR), 523 nm (CYPOR–iNOS^T), 528 nm (CYPOR-eNOS^T), and 530 nm (CYPOR-nNOS^T). The protein concentrations were 1 μM each, and reactions were performed as described in Materials and Methods.