Formation of two hydrogen bonds from the globin to theheme-linked oxygen molecule in Ascaris hemoglobin.

Abstract

We have tried to rind out why Ascaris hemoglobin has such an exceptionally high oxygen affinity (P5o (2) showed that this high affinity was due to an extremely slow dissociation (off rate, koff 0.0041/s-1), whereas the association rate (on rate, kon) was normal. By contrast, the affinity for carbon monoxide is similar to that of vertebrate hemoglobins. Synthetic chemistry has indicated no way of strengthening the Fe2+-02 bond by the equivalent of nearly 3 kcal/mol. The Fe2+{-2 bond is polar with partial Fe3+2-O character, whereas the Fe2+0-C bond is nonpolar. In consequence, the iron-bound oxygen, but not the carbon monoxide, accepts a hydrogen bond from the distal histidine in myoglobin and in the a subunit of human hemoglobin (in the , ( subunit Fe and 02 are too far apart) (3). In Ascaris hemoglobin the slow off rate of oxygen, in contrast to the normal offrate of carbon monoxide, suggested that the bound oxygen might be stabilized by an additional hydrogen bond from the globin.The amino acid sequence of Ascaris hemoglobin showed that glutamine takes the place of the distal histidine (4, 5).This could serve equally well as a hydrogen bond donor to the bound oxygen, but that bond would be no stronger than the bond from histidine. Initially, the sequence gave no clue to the possible nature of a second hydrogen bond donor.Such a clue did come from the observation of Olson and collaborators (6) that the replacement of leucine B10 (residue 29 of the polypeptide) in sperm whale myoglobin by phenylalanine raised its oxygen affinity 10-fold, because a hydrogen atom in the benzene ring of the phenylalanine made a favorable contact with the bound oxygen (6). In Ascaris hemoglobin this position is occupied by tyrosine. However, the myoglobin mutant B10 leucine -+ tyrosine (L -+ Y) had a lowered oxygen affinity, with an off rate 50 times higher than that of the wild type, suggesting that tyrosine B10 was unlikely to contribute the high oxygen affinity of Ascaris hemoglobin (7).Ascaris hemoglobin consists of eight identical subunits, each containing two myoglobin-like domains in tandem, followed by a repetitive sequence ofpolar residues suggestive of a "zipper" (4,8). Kloek et al. (9) cloned into an M13mpl9 vector (10). Site-directed mutagenesis was carried out by the dut, ung-method with E. coli BW313 (11). After the DNA sequences of these clones had been confirmed by single-stranded sequencing (12) using Sequenase (United States Biochemical), the Xba 1-HindI1 fragment was excised and cloned between the Xba I and HindIll sites of a T7 RNA polymerase expression vector, pRK172 (13). The BL21(DE3) strain was transformed with these vectors, and a large-scale culture was carried out at 370C in 2x TY medium containing ampicillin (100 pg/ml).Cells were harvested after induction with 1 mM isopropyl f3-D-thiogalactopyranoside for 3 hr and suspended in 10 mM 1594The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance wi...

Thermoglobin, Oxygen-avid Hemoglobin in a Bacterial Hyperthermophile

“…Mutation of glutamine to leucine or alanine in the A. suum heme domain results in ϳ5-and ϳ60-fold increases, respectively, in k O 2 (44). Mutation of tyrosine to leucine or phenylalanine produces an even larger ϳ200 -600-fold increase in k O 2 (44,45), resulting in an ϳ100-fold decrease in K O 2 (45). Thus, in A. suum Hb, the B10 tyrosine and E7 glutamine motif enhances O 2 binding, with the B10 tyrosine side chain having a dominant role in O 2 stabilization by electrostatic interactions.…”

Crystal Structure of the Dioxygen-bound Heme Oxygenase from Corynebacterium diphtheriae

“…As a result, k O 2 decreases from 14 s Ϫ1 for wild-type to 0.67 s Ϫ1 for the V68N mutant, and K O 2 increases 4-fold. Naturally occurring Ascaris suum Hb has an extremely low oxygen dissociation rate constant (k O 2 ϭ 0.004 s Ϫ1 ) due to two strong hydrogen bonds between distal pocket Tyr and Gln side chains and bound O 2 (53,57). For all three heme proteins, V68N Mb, Ascaris Hb, and HmuO, the multiple hydrogen bonds in the distal pocket preferentially stabilize bound O 2 and not CO. As a result, the M values (K CO /K O 2 ) for these proteins are very small, Յ 10, compared with those of mammalian Hbs (M ϭ 200 -400), Mbs (M ϭ 30 -40), and model hemes (M ϭ 1,000 -4,000) (Table II) (39,58).…”

Application of the paramagnetic dipole field for solution NMR active site structure determination in low‐spin, cyanide‐inhibited ferric hemoproteins

“…The Val(E7) Strong Distal H-bond by Tyr(B10). The monomeric Hbs from some nematodes/trematodes such as Ascaris suum (27), and Paraphistomum epiclitum, exhibit (28) extraordinarily slow O 2 off-rates and high O 2 affinities that have been attributed to very strong H-bond by an unusual Tyr(B10). While it has not been possible to obtain suitable crystals for HbO 2 , a crystal structure of Paraphistomum metHbH 2 O has been reported (31).…”