Correlation between methods was very high ( r = 0.9–1.0) for aspartate aminotransferase (AST), calcium, glucose, and uric acid high ( r = 0.7–0.89) for creatine kinase (CK), phosphorus, potassium, and total protein moderate ( r = 0.5–0.69) for globulin and low ( r = 0.3–0.49) for albumin and sodium. It is also possible that the distal site conformational change caused by the mutation alters the polarity of the heme environment and/or redox potential of the heme iron.To compare results of clinical biochemical analysis using an Abaxis VetScan bench-top analyzer with reagents specifically marketed for avian use and a Hitachi 911 analyzer, plasma (both methods) and whole blood (VetScan method) samples from 20 clinically healthy Hispaniolan Amazon parrots ( Amazona ventralis) were analyzed. In the NO-E318A mutant complex, there is no more ionic bridge in the distal site and thus a strong electron donating character of cysteine thiolate or NO may be manifested. In the NO-wild-type P450 1A2 complex, however, the iron reduction may be in part canceled out by electrons from both proximal and distal (even slightly) sites. The cysteine thiolate of the P450 1A2 proximal site should push electrons to the heme iron and facilitate the iron reduction. This postulate bridge is possible, although it does require postulation of an effectively low p K of Glu-318. On the other hand, the carboxyl group of Glu-318 may make a different ionic bridge consisting of Fe-N-O-H at P450 1A2 in the way of forming an improper Fe-N-O orientation to reduce the heme iron ( Fig. This special ionic bridge consisting of the Fe-N-O-H bond may facilitate the iron reduction in these NO-Fe complexes by forming a proper Fe-N-O orientation to push electrons. Also, a polar solvent will form an ionic bridge with NO and heme iron. Thus, effects of these flexible and ionic substrates on the K value will be less pronounced than other aromatic hydrocarbon substrates ( Table 2). L-Arg and N -hydroxy-L-Arg are rather flexible and have an ionic character in contrast to other hydrocarbon substrates studied here. In contrast, bindings of other larger hydrocarbons such as phenanthrene and dibenzanthracene, that have an extra benzene ring(s) on the side of the long axis, may further distort the less linearity of the Fe-N-O bond. It seems that 7-ethoxycoumarin and anthracene bindings at or near the distal site of heme may partially release this constraint, leading to a more linear Fe-N-O bond. Perhaps the Fe-N-O bond angle in P450 1A2 may not be 180°, and is slightly bent due to distal amino acid constraint, even without any substrates. Phenanthrene and dibenzanthracene markedly increased the K value by more than 8-fold ( Table 2). 7-Ethoxycoumarin and anthracene markedly decreased the K value by 3.4-fold and by more than 37-fold, respectively.

Based on these results, together with other spectral and kinetic data, it is suggested that the NO-ferric complex stability of P450, and perhaps of NOS, is largely ascribed to an ionic bridge between NO and the distal carboxyl group. Addition of 1,2:3,4-dibenzanthracene or phenanthrene almost abolished the mutation effect on the NO complex. We found that a mutation at Glu-318 to Ala in the putative distal site of P450 1A2, suggested to be important in the O activation of P450 reactions, markedly facilitates the reduction of the NO-ferric complex.

In the present study, NO bindings to cytochrome P450 1A2 (P450 1A2) distal mutants were studied in the presence of various substrates. The NO complex stability of the thiolate-coordinated hemoproteins, however, appeared irreconcilable with the strong electron-donating capability of the cysteine thiolate. Both NOS and P450 form stable nitric oxide (NO)-ferric heme complexes, whereas an NO-ferric heme complex of methemoglobin, that has an imidazole-coordinated heme active site, is easily reduced. Nitric oxide synthase (NOS) has a thiolate-coordinated heme active site similar to that of cytochrome P450 (P450). Glycobiology and Extracellular Matrices.

Enzyme plotting software deltagraph