S-Sulfohemoglobin and disulfide exchange: The mechanisms of sulfide binding by Riftia pachyptila hemoglobins

S-Sulfohemoglobin and disulfide exchange: The mechanisms of sulfide binding by Riftia pachyptila hemoglobins

^*Department of Biological Sciences and Marine Science Institute, University of California, Santa Barbara, CA 93106; ^§Laboratoire de Spectrométrie de Masse Bio-Organique, Université Louis Pasteur, Centre National de la Recherche Scientifique, Unité de Recherche Associée 31, Institut de Chimie, 1 Rue Blaise Pascal, 67008 Strasbourg Cedex, France, and ^†Equipe Ecophysiologie, UPMC, Centre National de la Recherche Scientifique, Institut National des Sciences de l’Univers, Station Biologique, BP 74, 29682 Roscoff Cedex, France

Communicated by George N. Somero, Stanford University, Pacific Grove, CA (received for review October 2, 1997)

Abstract

The deep sea hydrothermal tube worm Riftia pachyptila possesses a multihemoglobin system with three different extracellular hemoglobins (Hbs; V1, V2, and C1): two dissolved in the vascular blood, V1 and V2, and one in the coelomic fluid, C1. V1 consists of four heme-containing chains and four linker chains. The globin chains making up V2 and C1 are, with one exception, common to V1. Remarkably these Hbs are able to bind oxygen and sulfide simultaneously and reversibly at two different sites. Two of the globin chains found in these three Riftia Hbs possess one free Cys residue and for at least one of the globins, the b-Cys⁷⁵ is conserved among vestimentifera (Lamellibrachia sp.) and pogonophora (Oligobrachia mashikoi). By selectively blocking the free Cys with N-ethylmaleimide and using electrospray ionization mass spectrometry experiments, we show that these Cys residues are involved in sulfide binding by Riftia Hbs. Moreover, we also demonstrate that the larger V1 Hb can form persulfide groups on its linker chains, a mechanism that can account for the higher sulfide-binding potential of this Hb.

Footnotes

↵ ‡ To whom reprint requests should be addressed at present address: Department of Biological Sciences and Marine Science Institute, University of California, Santa Barbara, CA 93106. e-mail: zal{at}lifesci.lscf.ucsb.edu.
ABBREVIATIONS:

Hb,

hemoglobin;

NEM,

N-ethylmaleimide;

ESI-MS,

electrospray ionization mass spectrometry