ABSTRACT
The previously reported nitric oxide precursor [Mn(PaPy2Q)NO]ClO4 (1), where (PaPy2QH) is N,N-bis(2-pyridylmethyl)-amine-N-ethyl-2-quinoline-2-carboxamide, was used to investigate the interaction between NO and the protein truncated hemoglobin N (trHbN) from the pathogen Mycobacterium tuberculosis. Oxy-trHbN is exceptionally efficient at converting NO to nitrate, with a reported rate constant of 7.45 × 10(8) M(-1) s(-1) [Ouellet, H., et al. (2002) Proc. Natl. Acad. Sci. U.S.A. 99, 5902] compared to 4 × 10(7) M(-1) s(-1) for oxy-myoglobin [Eich, R. F., et al. (1996) Biochemistry 35, 6976]. This work analyzed the NO dioxygenation kinetics of wild type oxy-trHbN and a set of variants, as well as the nitrosylation kinetics for the reduced (red-trHbN) forms of these proteins. The NO dioxygenation reaction was remarkably insensitive to mutations, even within the active site, while nitrosylation was somewhat more sensitive. Curiously, the most profound change to the rate constant for nitrosylation was effected by deletion of a 12-amino acid dangling N-terminal sequence. The deletion mutant exhibited first-order kinetics with respect to NO but was zero-order with respect to protein concentration; by contrast, all other variants exhibited second-order rate constants of >10(8) M(-1) s(-1). trHbN boasts an extensive tunnel system that connects the protein exterior with the active site, which is likely the main contributor to the protein's impressive NO dioxygenation efficiency. The results herein suggest that N-terminal deletion abolishes a large scale conformational motion, in the absence of which NO can still readily enter the tunnel system but is then prevented from binding to the heme for an extended period of time.
