Single-turnover of nitric-oxide synthase in the presence of 4-amino-tetrahydrobiopterin: proposed role for tetrahydrobiopterin as a proton donor.

Tetrahydrobiopterin (BH4) is an essential cofactor of nitric-oxide synthase (NOS) that serves as a one-electron donor to the oxyferrous.heme complex. 4-Aminotetrahydrobiopterin (4-amino-BH4) is a potent inhibitor of NO synthesis, although it mimics all allosteric and structural effects of BH4 and exhibits comparable redox properties. We studied the reaction of reduced endothelial NOS oxygenase domain with O2 in the presence of 4-amino-BH4 at -30 degrees C by optical and electron paramagnetic resonance (EPR) spectroscopy. With Arg as the substrate, we observed a trihydropteridine radical with a corresponding heme species that was oxyferrous, with a Soret maximum at 428 nm and no EPR signal. With NG-hydroxy-l-arginine (NHA) no pterin radical appeared, whereas an axial ferrous heme.NO complex was formed. The corresponding optical spectra, with Soret bands at 417/423 nm, suggest that the proximal sulfur ligand is protonated. Accordingly, 4-amino-BH4 serves as a one-electron donor to Fe(II).O2 with both Arg and NHA, but the reaction cycle cannot be completed with either substrate. We propose that protonation of Fe(II)O2- is inhibited in the presence of 4-amino-BH4. With Arg, dissociation of O2- and binding of O2 yields Fe(II).O2 and a pteridine radical; with NHA, reaction of the substrate with heme-bound O2- eventually yields Fe(II).NO and reduced 4-amino-BH4. These results suggest that BH4 donates a proton to Fe(II).O2- during catalysis and that inhibition by 4-amino-BH4 may be due to its inability to support this essential protonation step.

Role of ribonucleotide reductase in regulation of cell cycle progression during and after exposure to moderate hypoxia.

Earlier studies have shown that the retinoblastoma protein (pRb) is involved in cell-cycle regulation under conditions of moderate hypoxia, which is the term we use to denote oxygen concentrations just above the lower level giving full respiration, ie. 1300 ppm O2. We have studied the cell cycle regulatory influence of varying levels of ribonucleotide reductase under moderate hypoxia in human cancer cells with either functional (T47D and T47DHU-res) or non-functional pRb due to expression of HPV18 E7 (HeLa S3). In this study we adapted a cell-line to hydroxyurea (T47DHU-res) resulting in an increased level of ribonucleotide reductase tyrosyl-radical that is not cell-cycle dependent. Under moderate hypoxic stress, pRb becomes dephosphorylated and rebound in the cell nucleus in all phases of the cell cycle, in contrast to its function under aerobic conditions where it binds only during early G1. We have shown in this paper that, upon reoxygenation, dephosphorylation and binding to the nucleus of pRb is reversible in T47D cells, although phosphorylation is delayed by more than 12 hours. As a result of the dephosphorylation of pRb, T47D cells are between 12 and 24 hours slower in reinitiating their S-phase progression than HeLa S3 cells (not containing functional pRb) following reoxygenation. However, with an increased level of ribonucleotide reductase tyrosyl-radical in the T47DHU-res cells, the S-phase progression after reoxygenation is reinitiated earlier, although the pRb status of these cells is the same as in T47D cells.