Heme Displacement Mechanism of CooA Activation: MUTATIONAL AND RAMAN SPECTROSCOPIC EVIDENCE

Mohammed Ibrahim,Robert L Kerby,Mrinalini Puranik,Ingar H Wasbotten,Hwan Youn,Gary P Roberts,Thomas G Spiro

doi:10.1074/jbc.m605568200

Mohammed Ibrahim, Robert L Kerby + Show 5 more

Open Access

https://doi.org/10.1074/jbc.m605568200

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Abstract

The heme-containing protein CooA of Rhodospirillum rubrum regulates the expression of genes involved in CO oxidation. CooA binds its target DNA sequence in response to CO binding to its heme. Activity measurements and resonance Raman (RR) spectra are reported for CooA variants that bind DNA even in the absence of CO, those in which the wild-type residues at the 121-126 positions, TSCMRT, are replaced by the residues AYLLRL or RYLLRL, and also for variants that bind DNA poorly in the presence of CO, such as L120S and L120F. The Fe-C and C-O stretching resonance Raman (RR) frequencies of all CooAs examined deviate from the expected back-bonding correlation in a manner indicating weakening of the Fe-His-77 proximal ligand bond, and the extent of weakening correlates positively with DNA binding activity. The (A/R) YLLRL variants have detectable populations of a 5-coordinate heme resulting from partial dissociation of the endogenous distal ligand, Pro-2. Selective excitation of this population reveals downshifted Fe-His-77-stretching RR bands, confirming the proximal bond weakening. These results support our previous hypothesis that the conformational change required for DNA binding is initiated by displacement of the heme into an adjacent hydrophobic cavity once CO displaces the Pro-2 ligand. Examination of the crystal structure reveals a physical basis for these results, and a mechanism is proposed to link heme displacement to conformational change.

Highlights

An increasing volume of research has revealed the ubiquity of heme sensor proteins [1, 2], which regulate a range of biological activities in response to changing levels of the gaseous molecules CO, NO, or O2
The crystal structure of the inactive CO-free form reveals that the DNA binding domains of the two monomers are different from each other, but both are strikingly rearranged when compared with the structure of the active form of the homologous cAMP receptor protein (CRP) protein (Fig. 1)
How is the transition from off to on induced by CO binding to the heme? In our earlier study we proposed that the primary impetus for the repositioning of the DNA binding domains was displacement of the heme toward an adjacent cavity that can be identified in the CooA crystal structure (Fig. 5) together with a complementary displacement of the C helices [10]

Summary

Introduction

An increasing volume of research has revealed the ubiquity of heme sensor proteins [1, 2], which regulate a range of biological activities in response to changing levels of the gaseous molecules CO, NO, or O2. CooA is homologous to another well studied transcription factor, cAMP receptor protein (CRP) (6 – 8), whose crystal structure has been determined in the cAMP-bound active state. The A chain has a bend between the C and D helices as in active CRP, but the domain orientation is completely different In both monomers of inactive CooA, the F helices are buried from solvent and should be inactive for DNA binding. The latter alternative is supported by previous kinetic measurements [17], indicating that CO-bound CooA is a mixture of forms that are “closed” and “open” with respect to CO dissociation and has recently been discussed more fully in Roberts et al [3]

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