Abstract
Kinetic traces were generated for the nanosecond and slower rebinding of photodissociated CO to trHbN in solution and in porous sol-gel matrices as a function of viscosity, conformation, and mutation. TrHbN is one of the two truncated hemoglobins from Mycobacterium tuberculosis. The kinetic traces were analyzed in terms of three distinct phases. These three phases are ascribed to rebinding: (i) from the distal heme pocket, (ii) from the adjacent apolar tunnel prior to conformational relaxation, and (iii) from the apolar tunnel subsequent to conformational relaxation. The fractional content of each of these phases was shown to be a function of the viscosity and, in the case of the sol-gel-encapsulated samples, sample preparation history. The observed kinetic patterns support a model consisting of the following elements: (i) the viscosity and conformation-sensitive dynamics of the Tyr(B10) side chain facilitate diffusion of the dissociated ligand from the distal heme pocket into the adjacent tunnel; (ii) the distal heme pocket architecture determines ligand access from the tunnel back to the heme iron; (iii) the distal heme pocket architecture is governed by a ligand-dependent hydrogen bonding network that limits the range of accessible side chain positions; and (iv) the apolar tunnel linking the heme site to the solvent biases the competition between water and ligand for occupancy of the vacated polar distal heme pocket greatly toward the nonpolar ligand. Implications of these finding with respect to biological function are discussed.
Highlights
Introduction to TrHbsTwo distinct groups of Hbs are readily distinguished within unicellular organisms [1]
The observed kinetic patterns support a model consisting of the following elements: (i) the viscosity and conformation-sensitive dynamics of the Tyr(B10) side chain facilitate diffusion of the dissociated ligand from the distal heme pocket into the adjacent tunnel; (ii) the distal heme pocket architecture determines ligand access from the tunnel back to the heme iron; (iii) the distal heme pocket architecture is governed by a ligand-dependent hydrogen bonding network that limits the range of accessible side chain positions; and (iv) the apolar tunnel linking the heme site to the solvent biases the competition between water and ligand for occupancy of the vacated polar distal heme pocket greatly toward the nonpolar ligand
In the following discussion of the kinetic patterns observed for trHbN, the kinetics will be dissected in terms of specific phases that can be directly related to events occurring on a molecular level
Summary
Two distinct groups of Hbs are readily distinguished within unicellular organisms [1]. The first one, occurring in bacteria and fungi, comprises the single chain flavohemoglobins that consist of an amino-terminal heme domain displaying the classical “myoglobin fold” and a carboxyl-terminal domain related to ferrodoxin reductases. The second group is the trHbs. The second group is the trHbs These are widely distributed in prokaryotes, unicellular eukaryotes, and plants, forming a distinct group within the Hb superfamily. Many occur in bacteria pathogenic to man. Three groups (groups I, II, and III) can be distinguished within the trHb family. TrHbN is a group I trHb found in M. tuberculosis
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