Role of tetrahydrobiopterin in biological NO synthesis

Nitric oxide synthase (NOS) catalyses the production of nitric oxide (NO). A cytochrome P450-like oxygenase, it uses two monooxygenation steps to convert L-arginine (L-arg) first to N -hydroxy-L-arginine (NOHA), a stable intermediate, and then to L-citrulline and NO. Mammalian NOSs are homodimeric enzymes. Each monomer is composed of an oxygenase domain (containing the L-arg binding site, a heme ligated by a cysteine thiolate, and a tetrahydrobiopterin (H4B)) and a reductase domain (binding NADPH, FAD, and FMN). NOS substrates are O2, L-arg, and NADPH. NADPH is the source of electrons required for oxygen activation. H4B is a vital cofactor that aids dimerisation and acts as a reducing/oxidising agent. Controversy still exists as to the final oxygenating species in the NOS mechanism, but the general reaction scheme is known. The ferric heme is reduced to the ferrous state by an electron from the reductase domain. Then oxygen binds to form the oxy-ferrous species. Then H4B donates an electron to form a peroxy-ferric species. It is likely this then forms a compound 1 (Fe(IV)+.=O) species that is the final oxygenating species. This thesis probes the mechanism of NOS to further define the mechanistic intermediates involved. The role of H4B in NO synthesis has been probed in both normal turnover conditions and special case reactions. To elucidate this mechanism further a mutant with a residue capable of stabilising the activated oxygen species was created, G586S, where glycine 586 of nNOS was replaced with a serine. This serine was within hydrogen bonding distance of the oxy-heme. A stabilised intermediate was observed by stopped flow reaction in the presence of H4B, but not aH4B (an inactive pterin analogue). Here single turnover reactions, each following either the reaction of L-arg to NOHA or NOHA to citrulline, were performed on the mutant using an external source of electrons. The reaction products were observed by HPLC. The mutant appears capable of the conversion of NOHA to citrulline, but not L-arg to NOHA. The WT enzyme appears capable of both. The intermediate is observed with either L-arg or NOHA bound, suggesting both reactions proceed via the same active oxygenating species. The inability of the mutant to catalyse the conversion of L-arg to NOHA may be due to protonation of the substrate hindering reaction such that the active oxygenating species decays before reaction can occur. This mutation, in allowing separation of the two monooxygenation steps, deserves further study. H4B binds at the dimer interface of NOS. Here the -systems of the pterins are only 13Å apart. This is within allowed distances for efficient electron transfer. Electron transfer between hemes, via the pterins, would allow a route for the breakdown of a dead end, ferrous-NO, species. Stopped flow monitoring of the decay of the ferrous-heme NO complex with nNOSoxy dimers with varying proportions of the hemes in the ferrous heme-NO complex showed no electron transfer between hemes of the dimer. The rate of decay of the ferrous heme-NO complex in oxygenated buffer is 0.12 s-1 for all conditions tested here. H4B-deficiency leads to several diseases. H4B makes a poor drug due to instability and cost, the search for druggable analogues of it is ongoing. H4B analogues blocked at the 6,7-positions in the dihydropterine-form have been screened here for catalytic activity. Several have shown comparable ability to catalyse NO production in vitro. Structure function analysis of these analogues has revealed the extent extension is tolerated at the C6 and C7 positions of the pterin

Blocked dihydropteridines as nitric oxide synthase activators

It has been shown that 6-acetyl-7,7-dimethyl-5,6,7,8-tetrahydropteridin-4(3H)-one can act as a competent cofactor for the production of nitric oxide by neuronal nitric oxide synthase (nNOS). More information was sought on the structural features that could contribute to strong binding within the enzyme whilst maintaining a fast electron transfer rate. This study was concerned with expansion at the C2-position of the pteridine scaffold. The evidence suggests that expansion at the C2-position had a deleterious effect with respect to Km and as a consequence electron transfer rate. Unexpectedly, several lines of evidence suggested that a methyl substituent on nitrogen at C2 reduced the electron density in the pyrimidine and dihydropterin rings

Tetrahydrobiopterin and electron transfer in NO synthase

Mammalian NO synthase requires the cofactor tetrahydrobiopterin (H4B) to act as an electron donor during the activation of molecular oxygen at the heme site. After donating an electron, the resultant H4B radical is then required to abstract an electron from the ferrous NO complex, which is generated at the end of the catalytic reaction, in order to facilitate NO release. We have recently explored the structural requirements of NO synthase for the H4B cofactor by studying a range of novel cofactor analogues with highly modified structures. Substituents on the C6 and C7 positions of H4B are tolerated well, with surprisingly bulky pterins being able to bind and drive NO synthesis. The modified pterins have a wide range of activities and binding constants, but the main function of the cofactors in activating molecular oxygen appears to be independent of C6 and C7 modification as shown by rapid reaction studies. We have also assessed the possibility of direct electron transfer across the dimer interface between H4B molecules in the two NO synthase subunits. The H4B cofactors are within the range for facile electron transfer and present a possible mechanism for NO synthase to escape from the unreactive ferrous-NO complex, which is known to originate from product inhibition