A Leucine Residue “Gates” Solvent but Not O2Access to the Binding Pocket of Phascolopsis gouldiiHemerythrin

Abstract

A leucine residue, Leu-98, lines the O 2 -binding pocket in all known hemerythrins. Leu-98 in recombinant Phascolopsis gouldii hemerythrin, was mutated to several other residues of varying sizes (Ala, Val), polarities (Thr, Asp, Asn), and aromaticities (Phe, Tyr, Trp). UVvisible and resonance Raman spectra showed that the di-iron sites in these L98X Hrs are very similar to those in the wild type protein, and several of the L98X hemerythrins formed stable oxy adducts. Despite the apparently tight packing in the pocket, all of the L98X Hrs except for L98W, had second order O 2 association rate constants within a factor of 3 of the wild type value. Similarly, the O 2 dissociation rate constant was essentially unaffected by substitutions of larger (Phe) or smaller (Val, Thr) residues for Leu-98. L98Y Hr showed a 170-fold decrease in the O 2 dissociation rate constant and a large D 2 O effect on this rate, which are attributed to a hydrogen-bonding interaction between the Tyr-98 hydroxyl and the bound O 2 . Significant increases in autoxidation rates were observed for all of the L98X Hrs other than X ‫؍‬ Tyr. These increases in autoxidation rates are attributed to increased solvent access to the binding pocket caused by inefficient packing (Phe), smaller size (Val, Ala), or increased polarity (Thr, Asp, Asn) of the residue 98 side chain. A leucine at position 98 appears to have the optimal size, shape, and hydrophobicity for inhibition of solvent access. Thus, "gating" of small molecule access to the binding pocket of Hr by Leu-98 is not evident for O 2 , but is evident for solvent.

A Bacterial Hemerythrin Domain Regulates the Activity of a Vibrio cholerae Diguanylate Cyclase

“…24,27,33,34 This conservation also extends to hydrophobic residues which line the O 2 binding pocket in invertebrate Hrs. An in vivo connection of Vc Bhr-DGC to iron is supported by the observation that, among the ~40 V. cholerae EL genes encoding GGDEF domain proteins, VC1216 is the only one whose transcription was found to be down-regulated under iron-depleted growth conditions.…”

Protein-Based Blood Substitutes: Recent Attempts at Controlling Pro-Oxidant Reactivity with and Beyond Hemoglobin

“…Hemerythrin (Hr) was shown to avoid reactivity towards hydrogen peroxide, nitric oxide and nitrite. Another advantage is the higher molecular weight than Hb (108 kDa vs 64 kDa), which should lead to lower levels of extravasation or elimination through the kidney [42,43]. Until now, glutaraldehyde-polymerized hemerythrin and polyethylene glycol-derivatized hemerythrin were successfully obtained.…”

“…Until now, glutaraldehyde-polymerized hemerythrin and polyethylene glycol-derivatized hemerythrin were successfully obtained. The effects of these chemical modifications on molecular weight, autooxidation rate and oxygen affinity appear to be favorable for blood substitute applications [42,43,44,45]. …”

Towards hemerythrin-based blood substitutes: Comparative performance to hemoglobin on human leukocytes and umbilical vein endothelial cells

“…Hemerythrin (Hr) is an oxygen carrier first described in marine invertebrates that employs a non-heme diiron active site (Farmer et al 2000;Jin et al 2002;Kryatov et al 2005). Hr was shown to avoid most of the stress-related sidereactions cited above for hemoglobin, showing much slower, if any, reactions with hydrogen peroxide, nitric oxide and nitrite (Kryatov et al 2005;Mot et al 2010).…”