Thr-E11 Regulates O2 Affinity in Cerebratulus lacteus Mini-hemoglobin

Abstract

The mini-hemoglobin from Cerebratulus lacteus (CerHb) belongs to a class of globins containing the polar Tyr-B10/Gln-E7 amino acid pair that normally causes low rates of O 2 dissociation and ultra-high O 2 affinity, which suggest O 2 sensing or NO scavenging functions. CerHb, however, has high rates of O 2 dissociation (k O 2 ‫؍‬ 200 -600 s ؊1 ) and moderate O 2 affinity (K O 2 Ϸ1 M ؊1 ) as a result of a third polar amino acid in its active site, Thr-E11. When Thr-E11 is replaced by Val, k O 2 decreases 1000-fold and K O 2 increases 130-fold at pH 7.0, 20°C. The mutation also shifts the stretching frequencies of both heme-bound and photodissociated CO, indicating marked changes of the electrostatic field at the active site. The crystal structure of Thr-E11 3 Val CerHbO 2 at 1.70 Å resolution is almost identical to that of the wildtype protein (root mean square deviation of 0.12 Å). The dramatic functional and spectral effects of the Thr-E11 3 Val mutation are due exclusively to changes in the hydrogen bonding network in the active site. Replacing Thr-E11 with Val "frees" the Tyr-B10 hydroxyl group to rotate toward and donate a strong hydrogen bond to the heme-bound ligand, causing a selective increase in O 2 affinity, a decrease of the rate coefficient for O 2 dissociation, a 40 cm ؊1 decrease in CO of hemebound CO, and an increase in ligand migration toward more remote intermediate sites.Globins are found in all kingdoms of living organisms. Their functions have been the subject of active debate. In addition to O 2 transport and storage (1-3), several novel functions have been proposed recently, including control of NO levels in microorganisms and nerve tissue, O 2 sensing, and dehaloperoxidase activity (4 -8). Nerve tissue Hbs are found in both vertebrates and invertebrates. Neuroglobin is a recently discovered member of the globin family, whose in vivo function is still unknown (9 -14). It is expressed in specific regions of vertebrate brains, displays low sequence identity to conventional Hbs or Mbs, 1 and is characterized by a bis-His-Fe hexacoordinate heme structure (11,12,15,16). In contrast, the nerve tissue Hbs found in mollusc, annelid, arthropod, nemertean, and nematode species (17, 18) appear to store and/or transport O 2 to support brain and axon function during temporary hypoxia (18 -21).The nerve tissue and body wall Hbs of the nemertean worm Cerebratulus lacteus (CerHb) 2 are the smallest naturally occurring Hbs, composed of only 109 amino acids. Analysis of the three-dimensional structure of nerve tissue CerHb has shown that the typical 3-over-3 globin fold is edited markedly (22). The N-terminal A-helix is deleted; the GH region is extended; and the C-terminal H-helix is shortened. Both sequence and fold comparisons suggest that CerHb is equally distant from all known globin tertiary structures, supporting its identification with a new superfamily, the mini-Hbs (22). CerHb contains a large elongated tunnel in its interior. Ligands may enter and exit CerHb through this apolar tunnel between th...

Thermoglobin, Oxygen-avid Hemoglobin in a Bacterial Hyperthermophile

“…In contrast, V. stercoraria Hb, in which the B10 tyrosine and E7 glutamine have not been observed to directly bind O 2 , has been shown to possess high O 2 affinity based on direct kinetic measurements of kЈ O 2 and k O 2 (54) (TABLE ONE). In the structure of the truncated neuronal Hb from the worm Cerebratulus lacteus, the B10 tyrosine and E7 glutamine participate in a hydrogen bond network with the E11 threonine that prevents stabilization of bound O 2 (55), and high O 2 affinity is generated only after mutation of the E11 threonine to valine (56 (2), supporting previous suggestions of a common origin (58,59). Because no single Hb class is widespread in all three domains, the identity of the common ancestor remains unclear.…”

The Apolar Channel in Cerebratulus lacteus Hemoglobin Is the Route for O2 Entry and Exit

“…Sample Preparation-Wild-type (wt) and mutant recombinant CerHbs were expressed and purified as described previously using a synthetic gene with codon usage optimized for expression in Escherichia coli (29,32). Most recombinant CerHb samples were isolated in the reduced state and used directly.…”

“…However, CerHb contains a Thr at the E11 position, and the Thr-48(E11) ␤-hydroxyl O-␥1 atom pulls the Tyr-11(B10) phenol hydrogen atom away from the bound ligand. As a result, the nonbonding electrons on the Tyr(B10) O-atom are pointing toward the bound ligand, and this negative partial charge causes the oxygen affinity of WT CerHb to be similar to that of vertebrate Mbs (K O2 Ϸ 1 M Ϫ1 ) (31,32). CerHb also displays very rapid rates of O 2 association (kЈ O2 ϭ 240 M Ϫ1 s Ϫ1 ) and dissociation (k O2 ϭ 180 s…”

“…These large rate coefficients seem surprising considering the crowded distal pocket and multiple hydrogen bonding interactions that appear to "tie" down the amide side chain of Gln-44, which forms the E7 gate in CerHb. Bolognesi and co-workers (29,32) have suggested that the large apolar channel between the E and H helices is the pathway for ligand movement into the heme pocket and accounts for the high rates of ligand binding and release. Thus, the loss of the A helix, which blocks access to the channel, may have been an adaptive strategy to allow for rapid rates of O 2 exchange while retaining the Tyr(B10)/Gln(E7) motif.…”

“…The mutant CerHb crystal structures were refined using REFMAC (45) and models inspected and modified using COOT (46 (12,37,38). The apparatus, method, and aspects of data collection have been described in general (39), and specific details for CerHbCO samples are presented in Pesce et al (32) and Deng et al (21). Briefly, the sample is cooled to an initial temperature at which the rebinding of CO is preferentially arrested on the experimental time scale of minutes to several hours (typically 3 K).…”