Effect of Heme Modification on Oxygen Affinity of Myoglobin and Equilibrium of the Acid−Alkaline Transition in Metmyoglobin

Abstract

Functional regulation of myoglobin (Mb) is thought to be achieved through the heme environment furnished by nearby amino acid residues, and subtle tuning of the intrinsic heme Fe reactivity. We have performed substitution of strongly electron-withdrawing perfluoromethyl (CF(3)) group(s) as heme side chain(s) of Mb to obtain large alterations of the heme electronic structure in order to elucidate the relationship between the O(2) affinity of Mb and the electronic properties of heme peripheral side chains. We have utilized the equilibrium constant (pK(a)) of the "acid-alkaline transition" in metmyoglobin in order to quantitatively assess the effects of the CF(3) substitutions for the electron density of heme Fe atom (rho(Fe)) of the protein. The pK(a) value of the protein was found to decrease by approximately 1 pH unit upon the introduction of one CF(3) group, and the decrease in the pK(a) value with decreasing the rho(Fe) value was confirmed by density functional theory calculations on some model compounds. The O(2) affinity of Mb was found to correlate well with the pK(a) value in such a manner that the P(50) value, which is the partial pressure of O(2) required to achieve 50% oxygenation, increases by a factor of 2.7 with a decrease of 1 pK(a) unit. Kinetic studies on the proteins revealed that the decrease in O(2) affinity upon the introduction of an electron-withdrawing CF(3) group is due to an increase in the O(2) dissociation rate. Since the introduction of a CF(3) group substitution is thought to prevent further Fe(2+)-O(2) bond polarization and hence formation of a putative Fe(3+)-O(2)(-)-like species of the oxy form of the protein [Maxwell, J. C.; Volpe, J. A.; Barlow, C. H.; Caughey, W. S. Biochem. Biophys. Res. Commun. 1974, 58, 166-171], the O(2) dissociation is expected to be enhanced by the substitution of electron-withdrawing groups as heme side chains. We also found that, in sharp contrast to the case of the O(2) binding to the protein, the CO association and dissociation rates are essentially independent of the rho(Fe) value. As a result, the introduction of electron-withdrawing group(s) enhances the preferential binding of CO to the protein over that of O(2). These findings not only resolve the long-standing issue of the mechanism underlying the subtle tuning of the intrinsic heme Fe reactivity, but also provide new insights into the structure-function relationship of the protein.

Insights Into How Heme Reduction Potentials Modulate Enzymatic Activities of a Myoglobin‐based Functional Oxidase

“…Thus, the two variants with high heme E°′ reveal 3‐fold lower O 2 affinity than parent F33Y‐Cu B Mb explaining why an increase in their ET rates does not translate directly to an increased O 2 reduction rates. Overall, these results indicate that increasing heme E°′ values leads to a decrease in O 2 affinity consistent with previous studies on Mb models that show electron‐withdrawing fluoro‐substituted hemes exhibiting low O 2 affinity values . This observation is further corroborated with R. sphaeroides cbb 3 oxidase that exhibits the lowest heme E°′ value of −59 mV and also displays the lowest K m for O 2 (7 n m ) among all oxidases .…”

“…Similarly,increasing hydrophobicity by addition of S92A residue close to catalytic heme iron in S92A-F33Y-Cu B Mb (K d = 0.2 AE 0.04 mm)i ncreases the O 2 affinity of heme by 3.5-fold. Finally,F 33Y-Cu B Mb (MF-heme) and F33Y-Cu B Mb (DFheme) exhibit rather weak K d values of 2.3 AE 0.4 mm and 2.0 AE 0.6 mm respectively.Thus,the two variants with high heme E8 8' [25] This observation is further corroborated with R. sphaeroides cbb 3 oxidase that exhibits the lowest heme E8 8' value of À59 mV and also displays the lowest K m for O 2 (7 nm)a mong all oxidases. [26] This apparent high O 2 affinity of cbb 3 oxidase helps them cope with extremely low concentration of O 2 (3-22 nm)i nr oot legumes.T hus,t uning heme E8 8' is an efficient method for HCOs to adapt to environmental constraints such as low O 2 concentration.…”

Tuning affinity and reversibility for O2binding in dinuclear Co(ii) complexes

“…For comparison, p(O 2 ) 50% for hemoglobin under physiological conditions has been found to be 35.0 × 10 −3 atm, and for myoglobin at 20 °C, p(O 2 ) 50% = 0.76 × 10 −3 atm. 27 Modification of the bridging carboxylato ligand provides a facile approach to tailor the oxygen affinity of dicobalt complexes of this type. A good measure for the electron-withdrawing ability of the carboxylato ligand is the pK a value of the parent acid, a Two complexes in the asymmetric crystallographic unit.…”

A Nuclear Resonance Vibrational Spectroscopic Study of Oxy Myoglobins Reconstituted with Chemically Modified Heme Cofactors: Insights into the Fe–O2 Bonding and Internal Dynamics of the Protein

“…The relationship between the ρ Fe and O 2 affinity could be interpreted in terms of the effect of a change in the ρ Fe on the equilibrium between ferrous-oxy (Fe 2+ −O 2 ) and ferric-superoxide (Fe 3+ −O 2 − ) states, and a decrease in the ρ Fe stabilizes the Fe 2+ −O 2 over Fe 3+ −O 2 − state, resulting in an increase of k off . 10 However, resonance Raman analysis of the MbO 2 s demonstrated that the conventionally assigned Fe−O 2 stretching band at ∼570 cm −1 of the protein was not largely affected by a change in the ρ Fe . 11 Thus, in order to examine the band assignment, we applied nuclear resonance vibrational spectroscopy (NRVS) 12−14 at SPring-8 15 to MbO 2 s reconstituted with 57 Fe-labeled Meso, Proto and 7-PF (MbO 2 (Meso), MbO 2 (Proto), and MbO 2 (7-PF), respectively) (Figure 1b).…”

“…Studies on Mbs reconstituted with chemically modified heme cofactors with variation of the electron density of the heme Fe atom (ρ Fe ) revealed that the O 2 affinity of the protein is regulated in such a manner that the O 2 affinity of the protein decreases, due to an increase in the O 2 dissociation rate constant ( k off ), with a decrease in the ρ Fe . The relationship between the ρ Fe and O 2 affinity could be interpreted in terms of the effect of a change in the ρ Fe on the equilibrium between ferrous-oxy (Fe 2+ –O 2 ) and ferric-superoxide (Fe 3+ –O 2 – ) states, and a decrease in the ρ Fe stabilizes the Fe 2+ –O 2 over Fe 3+ –O 2 – state, resulting in an increase of k off . However, resonance Raman analysis of the MbO 2 s demonstrated that the conventionally assigned Fe–O 2 stretching band at ∼570 cm –1 of the protein was not largely affected by a change in the ρ Fe .…”