NO Binding Induced Conformational Changes in a Truncated Hemoglobin from Mycobacterium tuberculosis

Abstract

The resonance Raman spectra of the NO-bound ferric derivatives of wild-type HbN and the B10 Tyr f Phe mutant of HbN, a hemoglobin from Mycobacterium tuberculosis, were examined with both Soret and UV excitation. The Fe-N-O stretching and bending modes of the NO derivative of the wild-type protein were tentatively assigned at 591 and 579 cm -1 , respectively. Upon B10 mutation, the Fe-NO stretching mode was slightly enhanced and the bending mode diminished in amplitude. In addition, the N-O stretching mode shifted from 1914 to 1908 cm -1 . These data suggest that the B10 Tyr forms an H-bond(s) with the heme-bound NO and causes it to bend in the wild-type protein. To further investigate the interaction between the B10 Tyr and the heme-bound NO, we examined the UV Raman spectrum of the B10 Tyr by subtracting the B10 mutant spectrum from the wild-type spectrum. It was found that, upon NO binding to the ferric protein, the Y 8a mode of the B10 Tyr shifted from 1616 to 1622 cm -1 , confirming a direct interaction between the B10 Tyr and the heme-bound NO. Furthermore, the Y 8a mode of the other two Tyr residues at positions 16 and 72 that are remote from the heme was also affected by NO binding, suggesting that NO binding to the distal site of the heme triggers a large-scale conformational change that propagates through the pre-F helix loop to the E and B helices. This large-scale conformational change triggered by NO binding may play an important role in regulating the ligand binding properties and/or the chemical reactivity of HbN.

Mycobacterial truncated hemoglobins: From genes to functions

“…These, together with a direct hydrogen bond from TyrCD1 to the ligand, lock the cyanide heme ligand in ferric M. tuberculosis trHbO (Milani et al, 2003). In keeping with these observations, resonance Raman data suggest that the heme-Fe-bound O 2 and CO are at hydrogen bonding distance to TyrB10, TyrCD1, and TrpG8 in ferrous M. tuberculosis trHbO (Mukai et al, 2002;Ouellet et al, 2003;Mukai et al, 2004). Stabilization of the heme-Fe-bound ligand by distal G8 and CD1 residues is unprecedented.…”

“…Comparable ligand stabilizing interactions (including residues TyrB10, GlnE11, and the ligand) are observed in the cyanide derivative of ferric M. tuberculosis trHbN, where the cyanide anion is bound in an orientation perpendicular to the heme plane (Milani et al, 2004b). Raman spectroscopy indicates that in M. tubercolosis trHbN the hemeFe-bound O 2 , CO, U NO, and OH -ligands are stabilized by hydrogen bonding to TyrB10 (Couture et al, 1999;Yeh et al, 2000;Mukai et al, 2002Mukai et al, , 2004.…”

“…The distinct features of the heme active site structure of U NO-responsive mycobacterial trHbs (see Milani et al, 2001;Visca et al, 2002a;Milani et al, 2003Milani et al, , 2004bMilani et al, , 2005 and their ligand binding properties (see Milani et al, 2005) combined with co-occurrence of multiple trHb classes in individual mycobacterial species and the temporal expression patterns of trHbs in vivo Fabozzi et al, 2006) suggest that these globins play different physiological functions, including scavenging of reactive nitrogen and oxygen species, O 2 uptake/transport, cellular respiration, and (pseudo-) enzymatic reactions. Interestingly, having retained only one trHb, M. leprae trHbO has been proposed to represent merging of multiple functions (Couture et al, 1999;Mukai et al, 2002;Ouellet et al, 2002;Pathania et al, 2002a;Visca et al, 2002a,b;Wittenberg et al, 2002;Ouellet et al, 2003;Liu et al, 2004;Mukai et al, 2004;Samuni et al, 2004;Milani et al, 2005;Ascenzi et al, 2006a,b;Fabozzi et al, 2006;Lama et al, 2006).…”

How Do Heme-Protein Sensors Exclude Oxygen? Lessons Learned from Cytochrome c′,Nostoc puntiformeHeme Nitric Oxide/Oxygen-Binding Domain, and Soluble Guanylyl Cyclase

“…Replacing His(E7) with apolar amino acids causes marked, 100-fold decrease in O 2 affinity of mutant Mbs and Hbs (70,80,98,111). In many bacterial and some invertebrate Hbs, a tyrosine at the B10 helical position serves as a strong Hbonding donor to preferentially stabilize bound O 2 , and in some of these cases, a glutamine at the E7 position further stabilizes the Fe(II)O 2 complex (16,30,38,45,76,89). In these proteins, replacement of TyrB10 by phenylalanine or GlnE7 by leucine markedly increases the O 2 -dissociation rate constant indicating loss of these favorable interactions (30,38,73,77).…”

Spectroscopic characterization of heme iron–nitrosyl species and their role in NO reductase mechanisms in diiron proteins

“…124 These stretching frequencies are at the low range of those reported for 6-coordinate heme {FeNO} 6 species with a proximal histidine ligand. 30,50,51,54,[126][127][128] Varotsis and coworkers 124 noted that the low ν(NO) contrasts with the high ν(CO) associated with inhibition of π-backbonding in the negative polarity of the distal pocket. 11 As discussed earlier (see section 2; spectroscopic signatures of heme iron-nitrosyl complexes), the influence of distal pocket environment on the characteristics of heme {FeNO} 6 species remains poorly understood.…”