NO formation by a catalytically self-sufficient bacterial nitric oxide synthase from Sorangium cellulosum

Abstract

The role of nitric oxide (NO) in the host response to infection and in cellular signaling is well established. Enzymatic synthesis of NO is catalyzed by the nitric oxide synthases (NOSs), which convert Arg into NO and citrulline using co-substrates O2 and NADPH. Mammalian NOS contains a flavin reductase domain (FAD and FMN) and a catalytic heme oxygenase domain (P450-type heme and tetrahydrobiopterin). Bacterial NOSs, while much less studied, were previously identified as only containing the heme oxygenase domain of the more complex mammalian NOSs. We report here on the characterization of a NOS from Sorangium cellulosum (both full-length, scNOS, and oxygenase domain, scNOSox). scNOS contains a catalytic, oxygenase domain similar to those found in the mammalian NOS and in other bacteria. Unlike the other bacterial NOSs reported to date, however, this protein contains a fused reductase domain. The scNOS reductase domain is unique for the entire NOS family because it utilizes a 2Fe2S cluster for electron transfer. scNOS catalytically produces NO and citrulline in the presence of either tetrahydrobiopterin or tetrahydrofolate. These results establish a bacterial electron transfer pathway used for biological NO synthesis as well as a unique flexibility in using different tetrahydropterin cofactors for this reaction.

Fig. 1.

Comparison of mammalian, bacterial, and S. cellulosum nitric oxide synthases. Shown is a schematic of the protein sequences indicating the N- and C-termini and the relative position of the identifiable domains. The heme domains are shown in gray, the reductase domains in white and the N terminus in mammalian NOS without sequence homology in the bacterial NOSs in black. In S. cellulosum NOS Fe binding Cys located between amino acid residues 479 and 516.

Fig. 2.

Decay of Fe(II)–O₂ and formation of Fe(III) in the presence of H₄B and Arg. Inset: Summary of the rates of Fe(II)–O₂ conversion into Fe(III) in scNOSox in the presence of various substrates and cofactors (see Materials and Methods for details).

Fig. 3.

Catalytic activity of scNOS. (A) Initial rates of NADPH and NADH consumption (monitored at A₃₄₀) and of NO synthesis (monitored at A₄₀₁ using the oxyhemoglobin assay) using reducing equivalents from NADPH and NADH. NO production rate was multiplied by 1.5 to account for the formal utilization of 1.5 NAD(P)H reducing equivalents required for the reaction. (B) Citrulline formation (end point measurement) in the oxyhemoglobin assay of scNOS activity. Experiments were performed in triplicate. Error bars represent standard deviations.

Fig. 4.

EPR analysis of scNOS. (A) EPR spectra of scNOS in the presence of Arg and H₄B with and without NADPH addition. (B) Experimental and simulated spectrum of the sample obtained after NADPH addition. All spectra were acquired at 9.4793 GHz.

Fig. 5.

Proposed electron transfer processes from NAD(P)H to heme via cofactors in the reductase domain.

Fig. 6.

Proposed mechanism of the chemical reactions at the heme with oxygen, substrates, and cofactors.