Effect of DNA Binding on Geminate CO Recombination Kinetics in CO-sensing Transcription Factor CooA

Abdelkrim Benabbas,Venugopal Karunakaran,Hwan Youn,Thomas L Poulos,Paul M Champion

doi:10.1074/jbc.m112.345090

Abstract

Carbon monoxide oxidation activator (CooA) proteins are heme-based CO-sensing transcription factors. Here we study the ultrafast dynamics of geminate CO rebinding in two CooA homologues, Rhodospirillum rubrum (RrCooA) and Carboxydothermus hydrogenoformans (ChCooA). The effects of DNA binding and the truncation of the DNA-binding domain on the CO geminate recombination kinetics were specifically investigated. The CO rebinding kinetics in these CooA complexes take place on ultrafast time scales but remain non-exponential over many decades in time. We show that this non-exponential kinetic response is due to a quenched enthalpic barrier distribution resulting from a distribution of heme geometries that is frozen or slowly evolving on the time scale of CO rebinding. We also show that, upon CO binding, the distal pocket of the heme in the CooA proteins relaxes to form a very efficient hydrophobic trap for CO. DNA binding further tightens the narrow distal pocket and slightly weakens the iron-proximal histidine bond. Comparison of the CO rebinding kinetics of RrCooA, truncated RrCooA, and DNA-bound RrCooA proteins reveals that the uncomplexed and inherently flexible DNA-binding domain adds additional structural heterogeneity to the heme doming coordinate. When CooA forms a complex with DNA, the flexibility of the DNA-binding domain decreases, and the distribution of the conformations available in the heme domain becomes restricted. The kinetic studies also offer insights into how the architecture of the heme environment can tune entropic barriers in order to control the geminate recombination of CO in heme proteins, whereas spin selection rules play a minor or non-existent role.

Highlights

We investigate the ultrafast dynamics of CO rebinding to different Carbon monoxide oxidation activator (CooA) complexes in order to better understand the activation mechanism of this very important class of proteins
Rebinding Kinetics of CO in RrCooA Complexes—Fluorescence anisotropy-based DNA-binding assays have shown that RrCooA binds to its target PcooF promoter sequence with high affinity, only in the presence of CO [11, 19]
In the presence of the PcooF sequence, the kinetics are faster by a factor of 2–3, and the fraction of the photodissociated CO that escapes into the solvent is reduced from about 4% to less than 2%

Summary

Introduction

Conclusion: DNA binding reduces heme heterogeneity and CO rebinding barrier This along with distal pocket trapping maintains the “on” state long enough for transcription to take place. The CO rebinding kinetics in these CooA complexes take place on ultrafast time scales but remain non-exponential over many decades in time. We show that this non-exponential kinetic response is due to a quenched enthalpic barrier distribution resulting from a distribution of heme geometries that is frozen or slowly evolving on the time scale of CO rebinding. Comparison of the CO rebinding kinetics of RrCooA, truncated RrCooA, and DNA-bound RrCooA proteins reveals that the uncomplexed and inherently flexible DNA-binding domain adds additional structural heterogeneity to the heme doming coordinate. The kinetic studies offer insights into how the architecture of the heme environment can tune entropic barriers in order to control the geminate recombination of CO in heme proteins, whereas spin selection rules play a minor or non-existent role

Methods

Results

Conclusion