Dataset Information

The pH dependence of heme pocket hydration and ligand rebinding kinetics in photodissociated carbonmonoxymyoglobin.

ABSTRACT: 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 His64 protonation and coupled protein conformational change.

SUBMITTER: Esquerra RM

PROVIDER: S-EPMC2376230 | biostudies-literature | 2008 May

REPOSITORIES: biostudies-literature

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The pH dependence of heme pocket hydration and ligand rebinding kinetics in photodissociated carbonmonoxymyoglobin.

Esquerra Raymond M RM Jensen Russell A RA Bhaskaran Shyam S Pillsbury Marlisa L ML Mendoza Juan L JL Lintner Benjamin W BW Kliger David S DS Goldbeck Robert A RA

The Journal of biological chemistry 20080320 20

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 ...[more]

PMID: 18359768

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Project description:Plant hemoglobins (Hbs) are found in nodules of legumes and actinorhizal plants but also in non-symbiotic organs of monocots and dicots. Non-symbiotic Hbs (nsHbs) have been classified into two phylogenetic groups. Class 1 nsHbs show an extremely high O2 affinity and are induced by hypoxia and nitric oxide (NO), whereas class 2 nsHbs have moderate O2 affinity and are induced by cold and cytokinins. The functions of nsHbs are still unclear, but some of them rely on the capacity of hemes to bind diatomic ligands and catalyze the NO dioxygenase (NOD) reaction (oxyferrous Hb + NO → ferric Hb + nitrate). Moreover, NO may nitrosylate Cys residues of proteins. It is therefore important to determine the ligand binding properties of the hemes and the role of Cys residues. Here, we have addressed these issues with the two class 1 nsHbs (LjGlb1-1 and LjGlb1-2) and the single class 2 nsHb (LjGlb2) of Lotus japonicus, which is a model legume used to facilitate the transfer of genetic and biochemical information into crops. We have employed carbon monoxide (CO) as a model ligand and resonance Raman, laser flash photolysis, and stopped-flow spectroscopies to unveil major differences in the heme environments and ligand binding kinetics of the three proteins, which suggest non-redundant functions. In the deoxyferrous state, LjGlb1-1 is partially hexacoordinate, whereas LjGlb1-2 shows complete hexacoordination (behaving like class 2 nsHbs) and LjGlb2 is mostly pentacoordinate (unlike other class 2 nsHbs). LjGlb1-1 binds CO very strongly by stabilizing it through hydrogen bonding, but LjGlb1-2 and LjGlb2 show lower CO stabilization. The changes in CO stabilization would explain the different affinities of the three proteins for gaseous ligands. These affinities are determined by the dissociation rates and follow the order LjGlb1-1 > LjGlb1-2 > LjGlb2. Mutations LjGlb1-1 C78S and LjGlb1-2 C79S caused important alterations in protein dynamics and stability, indicating a structural role of those Cys residues, whereas mutation LjGlb1-1 C8S had a smaller effect. The three proteins and their mutant derivatives exhibited similarly high rates of NO consumption, which were due to NOD activity of the hemes and not to nitrosylation of Cys residues.

Dataset Information

The pH dependence of heme pocket hydration and ligand rebinding kinetics in photodissociated carbonmonoxymyoglobin.

Publications

The pH dependence of heme pocket hydration and ligand rebinding kinetics in photodissociated carbonmonoxymyoglobin.

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