Analysis of Noncovalent Subassemblies. ESI-TOF-MS was used to investigate the presence and masses of noncovalent subassemblies within the C1 Hb of Riftia pachyptila. The molecule was subjected to conditions that produced 200-kDa dodecameric subassemblies in samples of various HBL Hbs (1). No subassemblies were detected in the spectra of the C1 Hb, reinforcing the idea that the C1 Hb is extremely stable. The mass of the intact C1 Hb was determined by ESI-TOF-MS to be 415.9 kDa. This mass is 1.0% higher than the mass of C1 (411.9 kDa) predicted as the sum of the masses of six A2 (b) monomers (16182.1 Da), six A1B2 (D2) dimers (32586 Da), six B1 monomers [comprising three (a) (17763.2 Da) and three (c) (16809.9 Da) monomers], 24 hemes (616.5 Da), and 12 Zn²⁺ ions (65.4 Da). The masses of the subunits used in this prediction were calculated as the intensity-weighted masses, including adducts observed in the spectrum of denatured C1. The mass of chain (a) was calculated as the intensity-weighted mean of glycosylated isoforms (a1), (a2), and (a3) observed in the spectrum of denatured C1. The mass excess of measured over predicted mass (1.0%) is broadly in line with previous work on various 200-kDa HBL subunits (1), where the mass excess varied up to 0.6% by using similar procedures.

The extensive contacts between dodecamers create a very tight molecular assembly, and the spherical shape of the multifunctional R. pachyptila C1 Hb provides a stable, compact platform for a large number of oxygen- and sulfide-binding sites. The closed point group D3 symmetry assures that no surfaces used for protein contacts are unsatisfied. Small heat shock proteins (sHSP) from Methanococcus jannaschii maintain a similar assembly. This methanogenic archaean expresses a 400-kDa sHSP with 24 subunits in response to temperature stress (2). It is believed that partially denatured proteins bind to the outer surface of the sHSP during heat stress, while the inner core of this molecule serves to protect essential enzymes from denaturation (2). Both M. jannaschii and R. pachyptila can be exposed to relatively high and variable temperatures within their respective environments (3). R. pachyptila can experience a significant difference in temperature over the length of its tube because of mixing of diffuse hydrothermal fluids and ambient deep-sea water (4). The large buried surface area between the polypeptide chains and dodecamers in the C1 Hb (2140 Ų between dimers) and 400-kDa sHSP (1558 Ų between dimers) (5) likely imparts greater stability during thermal stress. Protein stabilization such as this could especially benefit newly recruited tubeworms as they colonize nascent vent openings and are exposed to elevated temperature vent fluid, leaving the sea floor following weeks spent developing in water that is 2ºC (6).

1. Green, B. N., Gotoh, T., Suzuki, T., Zal, F., Lallier, F. H., Toulmond, A. & Vinogradov, S. N. (2001) J Mol Biol 309, 553-560.

2. Kim, R., Lai, L., Lee, H. H., Cheong, G. W., Kim, K. K., Wu, Z., Yokota, H., Marqusee, S. & Kim, S. H. (2003) Proc. Natl. Acad. Sci. USA 100, 8151-8155.

3. Jones, W. J., Stugard, C. E. & Jannasch, H. W. (1989) Arch. Microbiol. 151, 314-318.

4. Arp, A. J., Childress, J. J. & Fisher, C. R. (1985) Bull. Biol. Soc. Wash. 6, 289-300.

5. Kim, K. K., Kim, R. & Kim, S. H. (1998) Nature 394, 595-599.

6. Marsh, A. G., Mullineaux, L. S., Young, C. M. & Manahan, D. T. (2001) Nature 411, 77-80.