Altered Ligand Rebinding Kinetics Due to Distal-side Effects in Hemoglobin Chico (Lysβ66(E10) → Thr)

Self Cite

؊ as anions bind to lower affinity sites and shift the conformational equilibrium toward the R state. This pattern has been observed for various hemoglobins with a variety of small anions. Steric rather than electronic effects are invoked to explain the fact that no comparable reversal of oxygen affinity is observed under identical conditions. Evidence is presented to show that increases in hydrophilicity in the distal heme pocket can decrease oxygen affinity via steric hindrance effects while increasing the ease of anaerobic oxidation.The globin structure controls the redox potential of the heme site of hemoglobins (Hbs) 1 and myoglobins (Mbs), protects them from rapid oxidation, and thereby allows for reversible oxygen binding. Studying the redox properties of such systems allows us to gain insight on how anion-induced alterations fine tune the equilibrium between the oxidized and the reduced forms of Hbs. Our results extend earlier studies that have brought to light many possible modes of altering the electronic and ligand binding properties of the active site iron atoms (1-21).Although a large body of literature describes mechanisms involved in controlling the oxygen affinities of Hbs and Mbs, there are still unanswered questions with regard to how globin structure controls the redox potential (E1 ⁄2

Section: Resultsmentioning

confidence: 99%

Concentration-dependent Effects of Anions on the Anaerobic Oxidation of Hemoglobin and Myoglobin

Taboy¹,

Faulkner²,

Kraiter³

et al. 2000

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“…These decreases include the reactivity of the ␤93 cysteines (50), the on-rates for CO binding (51,52), T-state oxygen affinity (17,31,43,45,(51)(52)(53)(54)56), and the rate of T-state tertiary relaxation upon ligand binding (26). These results have been interpreted in terms of effector-induced damping of the amplitudes of those conformational dynamics that (i) transiently expose the sulfhydryl groups of Cys-␤93, (ii) transiently enhance ligand access to the heme iron (57), and (iii) facilitate ligand binding induced conformational relaxation (21). These earlier studies in combination with the present MEM results provide a basis for evaluating variations in reactivity within the T-state.…”

Section: Discussionmentioning

confidence: 99%

Nitrite Reductase Activity of Sol-Gel-encapsulated Deoxyhemoglobin

Roche

Dantsker

Samuni

et al. 2006

Nitrite reductase activity of deoxyhemoglobin (HbA) in the red blood cell has been proposed as a non-nitric-oxide synthase source of deliverable nitric oxide (NO) within the vasculature. An essential element in this scheme is the dependence of this reaction on the quaternary/tertiary structure of HbA. In the present work sol-gel encapsulation is used to trap and stabilize deoxy-HbA in either the T or R quaternary state, thus allowing for the clear-cut monitoring of nitrite reductase activity as a function of quaternary state with and without effectors. The results indicate that reaction is not only R-T-dependent but also heterotropic effector-dependent within a given quaternary state. The use of the maximum entropy method to analyze carbon monoxide (CO) recombination kinetics from fully and partially liganded sol-gel-encapsulated T-state species provides a framework for understanding effector modulation of T-state reactivity by influencing the distribution of high and low reactivity T-state conformations.The physiological role of the nitrite ion is currently attracting considerable research interest arising primarily from observations that indicate the anion can function as a non-nitric-oxide synthase source of nitric oxide (NO) (1-6). It has also been suggested that hemoglobin (Hb) 2 within red blood cells may be a source of deliverable nitrite-derived NO, generated from Hb nitrite reductase activity (deoxy-Hb ϩ nitrite 3 met-Hb ϩ NO) (6 -10). Such a mechanism also has clear implications for the vasoactivity of acellular hemoglobin-based blood substitutes. Although there are studies that support the physiological role of a hemoglobin-based source of nitrite-derived NO, there are still fundamental mechanistic questions that remain unanswered including the key issue of how NO, once bound to a ferrous heme, can be efficiently delivered (11, 12).Studies show that there is a relationship between the hemoglobin P50 and nitrite reductase activity (2, 3, 13, 14) as well as S-nitrosothiol synthase function (14). This result has led to the hypotheses that red blood cell/Hb enzymatic pathways can modulate blood flow and play a role in regulating hypoxic vasodilation (3, 14). The results also imply a relationship between the conformational properties of Hb and nitrite reductase activity (2, 5, 6, 15) and S-nitrosothiol synthase function (14). Direct measurements of the nitrite reductase activity of deoxy-Hb as a function of added allosteric effectors and mutagenic/chemical modifications support the concept of conformational control of Hb nitrite reductase reactivity with the T-state conformation having reduced reactivity compared with R-state conformations of Hb (5, 6). Two limitations of these studies are the inability to stabilize and compare the T and R-state forms of deoxy-HbA (human adult hemoglobin) without mutagenic or chemical modification and the complexity of the reductase reaction in solution due to autoacceleration arising from the allosteric nature of Hb reactivity.The present study directly addresses the qu...

“…The ␣ chains of the oxygen transport protein hemoglobin, for example, are known to contain a non-coordinated distal water molecule in the deoxy heme pocket that may modulate ligand affinities in a manner similar to that observed in myoglobin (10). Similarly, the human hemoglobin variant Hb Chico has a higher ␤-chain distal water occupancy than Hb A 0 , a factor that is thought to contribute to its lower oxygen affinity (11,12). The access of water molecules to the distal heme pockets of hemoglobins from the invertebrate Lucina pectinata (13) and the mycobacterium Mycobacterium tuberculosis (14) also appears to modulate ligand binding function in those proteins.…”

mentioning

confidence: 99%

The pH Dependence of Heme Pocket Hydration and Ligand Rebinding Kinetics in Photodissociated Carbonmonoxymyoglobin

Esquerra

Jensen

Bhaskaran

et al. 2008

We monitored the occupancy of a functionally important non-coordinated water molecule in the distal heme pocket of sperm whale myoglobin over the pH range 4.3-9.4. Water occupancy was assessed by using time-resolved spectroscopy to detect the perturbation of the heme visible band absorption spectrum caused by water entry after CO photodissociation (Goldbeck, R. A., Bhaskaran, S., Ortega, C., Mendoza, J. L., Olson, J. S., Soman, J., Kliger, D. S., and Esquerra, R. M. (2006) Proc. Natl. Acad. Sci. U. S. A. 103, 1254 -1259). We found that the water occupancy observed during the time interval between ligand photolysis and diffusive recombination decreased by nearly 20% as the pH was lowered below 6. This decrease accounted for most of the concomitant increase in the observed CO bimolecular recombination rate constant, as the lower water occupancy presented a smaller kinetic barrier to CO entry into the pocket at lower pH. These results were consistent with a model in which the distal histidine, which stabilizes the water molecule within the distal pocket by accepting a hydrogen bond, tends to swing out of the pocket upon protonation and destabilize the water occupancy at low pH. Extrapolation of this model to lower pH suggests that the additional increase in ligand association rate constant observed previously in stopped-flow studies at pH 3 may also be due in part to reduced distal water occupancy concomitant with further His 64 protonation and coupled protein conformational change.