NADPH- and NADH-nitrate reductases from soybean leaves.

Treatment of rabbit hemopexin with bromoacetic acid (BrAc) or with diethylpyrocarbonate (DEP) modified histidine residues and produced a concomitant decrease in the protein's ability to form a low-spin hemichrome complex with deuteroheme (ferrideuteroporphyrin IX). Deuteroheme bound to hemopexin before treatment decreased the extent of inactivation by either reagent. After exposure of deuteroheme-hemopexin to 0.16 m BrAc at pH 6.9 for 120 h, 10–11 of the 16 histidine residues of hemopexin were carboxymethylated, but 90–95% of the deuteroheme-hemopexin complex remained intact. Under the same conditions, 12 histidine residues of apo-hemopexin were carboxymethylated, and 95% of the protein's ability to form its normal hemichrome complex with heme (ferriprotoporphyrin IX) was abolished. The alkylated apo-protein, however, did retain a potential to interact with deuteroheme. The apparent dissociation constants for the complexes of metal-free deuteroporphyrin and deuteroheme with BrAc-treated apo-hemopexin were both about 10^?6m and nearly equal to that of the native deuteroporphyrin-hemopexin complex, as assessed by quenching of tryptophan fluorescence.Approximately 10 histidyl residues of the deuteroheme-hemopexin complex, but only about 4 residues of the apo-protein, were modified by DEP before heme-binding was appreciably affected. The effects of DEP on hemopexin were reversed by hydroxylamine at neutral pH, indicating that ethoxyformylation of histidine residues caused the observed inactivation of hemopexin. This and the results of BrAc treatment suggest that hemopexin contains several easily accessible histidine residues which are not critical for its interaction with heme.The conformation-sensitive positive ellipticity at 231 nm of hemopexin was affected by carboxymethylation and ethoxyformylation. Treatment with BrAc had only a small effect on the intrinsic ellipticity of apo-hemopexin, but eliminated the increase in ellipticity produced by interaction of unmodified hemopexin with heme. Treatment with DEP, on the other hand, decreased both intrinsic and extrinsic ellipticity.These results provide further evidence that the heme-hemopexin complex involves histidyl-heme iron coordination. In addition, they show that formation of the histidyl-heme complex not only greatly enhances the strength of the heme-hemopexin interaction but also is important for triggering conformational changes in the protein.