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Two oxygenated iron-cobalt hybrid hemoglobins (Hbs), (alpha Co-O2 beta Fe-O2)2 and (alpha Fe-O2 beta Co-O2)2, were studied by electron spin echo envelope modulation (ESEEM) spectroscopy in order to measure (i) electron-nuclear hyperfine and nuclear quadrupole coupling to the N epsilon of the proximal histidyl imidazole and (ii) nuclear hyperfine coupling to exchangeable 2H in the oxyCo subunits. 14N couplings were found to be smaller in the oxyCo alpha subunits than in the oxyCo beta subunits, suggesting a more ionic and shorter Co-O2 bond in the alpha subunits [Lee et al. (1994) Biochemistry 33, 7609], which correlates with the higher O2 affinity found for (alpha Co beta Fe-O2)2 Hb than for (alpha Fe-O2 beta Co)2 Hb [Imai et al. (1980) J. Mol. Biol. 138, 635]. A smaller nuclear quadrupole coupling constant found for the proximal histidyl N epsilon in the oxyCo alpha subunits also suggests an increase in the overlap between the N epsilon sp2 hybrid and the Co dz2 orbital, i.e., a shorter Co-N epsilon bond, than in the oxyCo beta subunits. On the other hand, the relative orientation of the g and 14N epsilon nuclear quadrupole tensors, obtained by spectral simulation, suggests that the Co-O-O bond angle is similar in the two types of oxyCo subunits. An X-ray crystallographic study of oxyFe Hb A [Shaanan, B. (1982) Nature 296, 683] has also reported similar Fe-O-O bond angles in both alpha and beta subunits, but with shorter Fe-N epsilon and Fe-O2 bonds in the alpha subunits.(ABSTRACT TRUNCATED AT 250 WORDS)

Oxidation of hemoglobins leads to the formation of Fe(III) forms, whose relevance spans from biochemical to physiological aspects 1. Here we report a combined EPR/X-ray crystallography study at acidic pH on six ferric tetrameric hemoglobins (Hbs), five isolated from the Antarctic fish species Trematomus bernacchii, Trematomus newnesi, and Gymnodraco acuticeps, and one from the sub-Antarctic fish Cottoperca gobio. Our data reveal that, in all the studied Hbs, aquomet form and two hemichromes coexist at acidic pH, analogously to what happens at physiological pH 2. Nevertheless, the EPR analysis reveals that, only for the three Root effect hemoglobins 3,4, a significant amount of pentacoordinated (5C) high-spin Fe(III) species appears at acidic pH. The crystal structure at acidic pH of the ferric hemoglobin from Trematomus bernacchii, endowed with Root effect 4, is also reported at 1.7 Å. Indeed, this structure reveals a 5C state both at α- and β-chains within a T-like quaternary structure. The ability of the Antarctic fish Hbs to form 5C states in various conditions prompted us to perform a peroxidase activity study. These proteins exhibit a peroxidase activity higher than that observed for mammalian and temperate fish Hbs , thus suggesting that a partial hemichrome state in tetrameric Hbs does not protect them from peroxidation as previously proposed 5. This work was financially supported by PNRA (Italian National Programme for Antarctic Research) 1 A. Vergara, L. Vitagliano, G. di Prisco, C. Verde, and L. Mazzarella, Meth. Enz. 436A, 421 (2008). 2 A. Vergara, M. Franzese, A. Merlino, L. Vitagliano, G. di Prisco, C. Verde, H. C. Lee, J. Peisach, and L. Mazzarella, Biophys. J. 93, 2822 (2007). 3 L. Mazzarella, G. Bonomi, M. C. Lubrano, A. Merlino, A. Vergara, L. Vitagliano, C. Verde, and G. di Prisco, Proteins: Struct. Funct. Bioinf. 62, 316 (2006). 4 L. Mazzarella, A. Vergara, L. Vitagliano, A. Merlino, G. Bonomi, S. Scala, C. Verde, and G. di Prisco, Proteins: Struct. Funct. Bioinf. 65, 490 (2006). 5 L. Feng, S. Zhou, L. Gu, D. Gell, J. Mackay, M. Weiss, A. Gow, and Y. Shi, Nature 435, 697 (2005).

Antarctic fish hemoglobins (AFHbs) exhibit a peculiar oxidation process. Upon oxidation, these tetrameric proteins show a remarkable propensity to evolve toward the formation of hemichrome, a species in which both proximal and distal hystidines are bound to the heme iron [1,2]. The AFHb isolated from Trematomus bernacchii also shows a drastic reduction of oxygen affinity and cooperativity at acidic pH ( Root effect), ascribed to an overstabilization of the T quaternary structure [3]. On the other hand, the AFHbs isolated from Trematomus newnesi , which bears a very high sequence identity with the former Hb, has a nearly pH-independent oxygen affinity. The crystal structure of the oxidized form of these two AFHbs has shown that a) the a chains go to aquo-met, whereas the b chains form the hemichrome, b) the quaternary structure is intermediate between the canonical R and T states [1,2]. The EPR analysis of the ferric AFHbs at physiological pH reveals two distinct hemichromes (I, II) in solution [4]. Interestingly, the pH dependence of EPR spectra and of the X-ray crystal structure of ferric HbTb suggests a correlation between the hemichrome stability and the Root effect. Indeed, the high-resolution crystal structures of the oxidized form of the AFHbs isolated from T. bernacchii at pH 7.6 and 6.0 (1.5 and 1.8 Å, respectively) show different quaternary structures and different heme coordination: at pH 7.6 the hemoglobin is in the a-aquomet/b-hemichrome form ad its quaternary structure is intermediate between the R and T state, whereas at pH 6 both hemes are penta-coordinated and the hemoglobin adopts the T quaternary structure. The EPR spectra of Root effect Hbs confirm the presence of a penta-coordinated, unligated ferric state at pH 6. Altogether, these findings suggest that the hemichrome instability at acidic pH for Root effect Hbs is related to some incompatibility of the hemichrome in the b subunits within a T quaternary structure. Interestingly, ferric AFHbs reveal a high peroxidasic activity with respect to mammalian and temperate fish hemoglobins. [1] Riccio A., et al. Proc. Natl. Acad. Sci. US, 2002, 99, 9801; [2] Vitagliano L., et al. Eur. .J. Biochem, 2004, 271, 1651. [3] Mazzarella L., et al. Proteins 2006, 65, 490. [4] Vergara A, et a., submitted.

Antarctic fish hemoglobins (AF-Hbs) exhibit an unusual auto-oxidation process. Our previous crystallographic and spectroscopic investigations have demonstrated that, upon oxidation, these tetrameric proteins show a remarkable propensity to evolve towards the formation of low-spin bis-histidine complexes, hemichromes, even under physiological conditions [1,2]. The crystal structures of the fully oxidized forms of Trematomus newnesi and Trematomus bernacchii AF-Hbs have shown that α and β chains follow different oxidation pathways. Indeed, α chains form aquo-met, whereas β chains form hemichromes. Interestingly, the quaternary structures of these ferric forms are intermediate [1,2] between the physiological R and T hemoglobin states [3]. Frozen solution EPR spectra of the ferric AF-Hbs at physiological pH reveal two distinct hemichrome signals (I, II), corresponding to two bis-histidine structures with different tilt angles, one of which (I) is consistent with the high resolution crystal structure (1.5 Å) of the oxidized form of T. bernacchii AF-Hb, HbTb. EPR studies show that one of the hemichromes (I) preferentially reacts with CN¯. With excess CN¯, a cyano-met and a rhombic high spin signal were resolved. These signals may correlate with the high resolution crystal structure of the ferric form of HbTb after cyanidation (1.4 Å), which exhibits two different β heme structures, attributed to a cyano-met and a penta-coordinated form. A possible functional role of hemichromes in Antarctic fish is discussed in terms of protection from oxidative stress [4].[1] Riccio A., Vitagliano L., Zagari A., di Prisco G., Mazzarella L. Proc. Natl. Acad. Sci. US, 2002, 99, 9801; [2] Vitagliano L., Bonomi G., Riccio A., di Prisco G., Smulevich G., Mazzarella L. Eur. .J. Biochem, 2004, 271, 1651. [3] Mazzarella L., Vergara A., Vitagliano L., Merlino A., Bonomi G., Scala S., Verde C., di Prisco G. Proteins, Str. Funct. Bioinf. in press. [4] Feng L., Zhou S., Gu L., Gell D.A., Mackay J. P., Weiss M.J., Gow A.J., Shi Y., Nature 2005, 435, 697.

The structural and spectrochemical effects of the replacement of Met44 in the hydrophobic surface patch of azurin from Pseudomonas aeruginosa by a lysine residue were studied as a function of the ionization state of the lysine. In the pH range 5-8, the optical absorption, resonance Raman, EPR and electron spin-echo envelope modulation spectroscopic properties of wild-type and Met44-->Lys (M44K) azurin are very similar, indicating that the Cu-site geometry has been maintained. At higher pH, the deprotonation of Lys44 in M44K azurin (pK(a) 9-10) is accompanied by changes in the optical-absorption maxima (614 nm and 450 nm instead of 625 nm and 470 nm) and in the EPR g(parallel-to) value (2.298 instead of 2.241), indicative of a change in the bonding interactions of Cu at high pH. The strong pH dependence of the electron self-exchange rate of M44K azurin supports the assignment of Lys44 as the ionizable group and demonstrates the importance of the hydrophobic patch for electron transfer. The pH dependence of the midpoint potentials of wild-type and M44K azurin can be accounted for by the ionizations of His35 and His83 and by the additional electrostatic effect of the mutation.