Heme d formation in a Shewanella benthica hemoglobin

Area of Science:

• Biochemistry
• Structural Biology
• Microbial Physiology

Background:

• Microbial globins, particularly truncated hemoglobins (HbN), play crucial roles in oxygen transport and metabolism in diverse bacteria.
• Shewanella benthica is an obligate psychropiezophile, adapted to extreme cold and high-pressure environments, with its HbN (SbHbN) structure being of interest.
• The active site of globins, specifically the heme environment, dictates their biochemical properties and potential for cofactor modification.

Purpose of the Study:

• To elucidate the structural and biochemical properties of truncated hemoglobin from Shewanella benthica (SbHbN).
• To investigate the reactivity of the heme cofactor within the SbHbN active site, particularly under oxidative conditions.
• To explore the potential for novel heme cofactor formation mediated by the globin fold.

Main Methods:

• Protein expression and purification of SbHbN.
• Crystallization of ferric SbHbN and X-ray crystallography to determine its structure.
• In vitro biochemical assays involving hydrogen peroxide treatment and site-directed mutagenesis (Tyr34Phe replacement).

Main Results:

• Crystallization of SbHbN revealed a unique heme d derivative with a γ-spirolactone and hydroxyl group, formed over weeks.
• In solution, hydrogen peroxide treatment led to heme d formation and covalent attachment of heme to the protein.
• The Tyr34Phe mutation abolished the formation of heme d and the heme-protein covalent linkage, highlighting Tyr34's critical role.

Conclusions:

• SbHbN exhibits remarkable reactivity, readily modifying its heme b cofactor into novel derivatives like heme d.
• The tyrosine residue at position 34 (Tyr34) is essential for mediating heme modification and covalent adduct formation.
• This study demonstrates the globin fold's capacity for supporting diverse heme chemistries, potentially leading to novel cofactor discovery.