Characterization of Variants Altered at the N-terminal Proline, a Novel Heme-Axial Ligand in CooA, the CO-sensing Transcriptional Activator

Abstract

CooA, the carbon monoxide-sensing transcription factor from Rhodospirillum rubrum, binds CO through a heme moiety resulting in conformational changes that promote DNA binding. The crystal structure shows that the N-terminal Pro 2 of one subunit (Met 1 is removed post-translationally) provides one ligand to the heme of the other subunit in the CooA homodimer. To determine the importance of this novel ligand and the contiguous residues to CooA function, we have altered the N terminus through two approaches: site-directed mutagenesis and regional randomization, and characterized the re- The sensing of dissolved gas molecules by proteins in biology has recently attracted considerable biochemical interest. The role of nitric oxide in a variety of important biochemical processes (1, 2), and its receptor, soluble guanylyl cyclase (sGC) 1 have been well documented in eukaryotic systems (3, 4). FixL, which modulates the expression of genes responsible for nitrogen fixation in rhizobia, is an example of an oxygen sensor (5, 6). An oxygen sensor in Escherichia coli, termed DOS ("direct oxygen sensor"), has been reported although its physiological role remains undefined (7). Finally, for carbon monoxide (CO), CooA, the CO-oxidation activator protein, modulates the expression of genes required for the utilization of CO as a sole energy source in the photosynthetic bacterium Rhodospirillum rubrum (8). All of the above mentioned proteins have in common a heme prosthetic group to which their respective gas molecules bind. The binding event is then followed by a conformational change in the protein that effects activity.Numerous studies have clearly demonstrated the physiological importance of CO in a wide variety of processes (9 -11), and although sGC has been implicated in sensing CO (12-14), direct evidence of a CO-receptor in eukaryotic signal transduction systems is lacking. CooA senses CO through a heme moiety and represents the current model system for biological CO-sensing (19,20). Finally, the ligand that is displaced upon binding CO remains speculative.Recently, the three-dimensional structure of Fe II CooA has been solved by x-ray diffraction techniques (21). This report showed that the general folding topology of CooA was indeed similar to that of CRP (22). In addition to the verification of His 77 as one of the heme-axial ligands in Fe II CooA, inspection of the structure identified the other axial ligand as an Nterminal proline residue (Pro 2 ; Met 1 is removed by processing) from the other subunit of the dimer. This structural environment represents an unprecedented axial ligation arrangement for a heme protein.In a previous study (18), we altered His 77 and found that the UV-visual spectra of these variants was normal in the Fe ʈ To whom correspondence should be addressed. Tel.: 608-262-3567; Fax: 608-262-9865; E-mail: groberts@bact.wisc.edu.1 The abbreviations used are: sGC, soluble guanylyl cyclase; CO, carbon monoxide; CRP, cAMP receptor protein; FixL, oxygen sensor of Rhizobium meliloti; Mb, myoglobin; P-4...

The Heme Pocket Afforded by Gly117 Is Crucial for Proper Heme Ligation and Activity of CooA

“…Recently, the x-ray crystal structure of Fe(II) CooA was solved to 2.6-Å resolution and revealed that Pro 2 of one monomer serves as one ligand to the heme iron of the other monomer and that the ligand trans to Pro 2 is His 77 (8). Previous studies, including electron paramagnetic resonance (EPR) 1 analysis and site-directed mutagenesis showed that one of the ligands in the Fe(III) form is Cys 75 in the thiolate form (7,9), and Pro 2 is assumed to be the trans ligand, based on indirect evidence (10,11). Thus, there is an unusual ligand switch upon reduction of the heme, with His 77 replacing Cys 75 (7,12).…”

Mapping CooA·RNA Polymerase Interactions

“…Fluorescence Polarization Assay for DNA Binding-In vitro DNA binding of CooA preparations was measured using a fluorescence polarization assay as described (28,29).…”

“…Although a comparison of the structure of effectorbound CRP with effector-free CooA shows a significant repositioning of the AR3 region (8) (Fig. 1), (28,30). There is strong reason to believe that CO binding perturbs the position of the heme 6 and, therefore, His 77 , Asn 42 , and Glu 41 residues as well.…”

“…CooA-This study began as a screen for mutations in cooA that would suppress mutations that alter His 77 , one of the normal heme ligands in the Fe II (8) and Fe II ϩ CO forms (28,30). The CooA variants in the starting strains (with either H77K 3 or H77E substitutions) have a very low level of COindependent activity and, although they bind CO with an affinity roughly similar to that of WT CooA, 4 CO binding does not result in increased activation of transcription (10).…”

Azurin–DNA Conjugate with the Binding Motif of a Transcriptional Regulator, CooA: CO-dependent Modulation of the Electron-transfer Reaction

“…In order to examine the specific association of CooA to Azu DNA in a CO-dependent manner, we carried out a gel mobility assay with Azu DNA or pristine ds-oligo-DNA containing CooA binding sequence (27 bp) 16 (Figure 2a). As previously reported, CO CooA caused a distinctive band shift of the pristine ds-oligo-DNA (Lane 3), 21 which was not observed for CooA in the CO-unbound form (Lane 2).…”