Robert W. Clark

Guanidine Hydrochloride-Induced Unfolding of the Three Heme Coordination States of the CO-Sensing Transcription Factor, CooA

CooA is a heme-dependent CO-sensing transcription factor that has three observable heme coordination states. There is some evidence that each CooA heme state has a distinct protein conformation; the goal of this study was to characterize these conformations by measuring their structural stabilities through guanidine hydrochloride (GuHCl) denaturation. By studying the denaturation processes of the Fe(III) state of WT CooA and several variants, we were able to characterize independent unfolding processes for each domain of CooA. This information was used to compare the unfolding profiles of various CooA heme activation states [Fe(III), Fe(II), and Fe(II)-CO] to show that the heme coordination state changes the stability of the effector binding domain. A mechanism consistent with the data predicts that all CooA coordination states and variants undergo unfolding of the DNA-binding domain between 2 and 3 M GuHCl with a free energy of unfolding of approximately 17 kJ/mol, while unfolding of the heme domain is variable and dependent on the heme coordination state. The findings support a model in which changes in heme ligation alter the structural stability of the heme domain and dimer interface but do not alter the stability of the DNA-binding domain. These studies provide evidence that the domains of transcription factors are modular and that allosteric signaling occurs through changes in the relative positions of the protein domains without affecting the structure of the DNA-binding region.

DNA binding by an imidazole-sensing CooA variant is dependent on the heme redox state

CooA is a transcription factor from Rhodospirillum rubrum that is regulated by the binding of the small molecule effector, CO, to a heme moiety in the protein. The heme in CooA is axially ligated by two endogenous donors in the Fe(III) and Fe(II) states of the protein, and CO binding to the Fe(II) state results in replacement of the distal ligand. Reduction of the heme in the absence of CO results in a ligand switch on the proximal side, in which a cysteine thiolate in the Fe(III) state is replaced by a histidine in the Fe(II) state. Recently, a variant, termed RW CooA, was designed to respond to a new effector; Fe(II) RW CooA shows high specificity and induced DNA-binding activity in the presence of imidazole. Spectroscopic characterization of the imidazole adducts of RW CooA revealed that, unlike CO, imidazole binds to both Fe(III) RW CooA and Fe(II) RW CooA. The spectral characteristics are consistent with normal function of the redox-mediated ligand switch; Fe(III)–imidazole RW CooA bears a thiolate ligand and Fe(II)–imidazole RW CooA bears a neutral donor ligand. Since the effector binds to both redox states, RW CooA was used to probe the role of the redox-mediated ligand switch in the CooA activation mechanism. Functional studies of Fe(III)–imidazole and Fe(II)–imidazole ligated RW CooA demonstrate that only the Fe(II)–imidazole form is active for DNA binding. Thus, the ligand switch is essential for the activating conformational change and may prevent aberrant activation of CooA by other neutral diatomic molecules.

Modeling proline ligation in the heme-dependent CO sensor, CooA, using small-molecule analogs.

CooA, the only protein known to employ proline as a heme ligand, is a CO-activated transcription factor found in the bacterium Rhodospirillum rubrum. Proline is a heme ligand in both the Fe(III) and Fe(II) states; the sixth ligand is cysteinate in Fe(III) CooA and histidine in Fe(II) CooA. When CO binds to Fe(II) CooA, it selectively replaces the proline ligand, activating the protein. The proposed roles of proline are to stabilize the heme pocket during the redox-mediated ligand switch and to form a weak metal–ligand bond that is preferentially cleaved to bind CO. To explore this latter proposal, binding affinity, structural, and density functional theory computational studies were performed using pyrrolidine and 2-methylpyrrolidine as analogs of proline, and imidazole as an analog of histidine. Measurement of the binding properties of these amino acid analogs in two different protein environments, CooA variant ΔP3R4 and myoglobin, revealed that CooA is tailored to accept the bulky proline ligand. Furthermore, the high pKa of proline facilitates selective replacement by CO. Model metalloporphyrin X-ray and computational structures suggest that the key factor leading to lengthening of the Fe–ligand bond and decreased binding affinity is steric hindrance at the C-2 position of the pyrrolidine ring. These data afford a more complete understanding of how CooA utilizes the weak proline ligand to direct CO to the distal position, thus ensuring selective retention of the histidine ligand.

A 1,2,4-diazaphospholane complex of rhodium.

The crystal structure of a prospective olefin catalyst, namely [2-[1-acetyl-5-(2-hydroxyphenyl)-4-phenyl-1,2,4-diazaphospholan-3-yl]phenyl acetate-kappaP]chloro(eta(4)-cycloocta-1,5-diene)rhodium(I) dichloromethane solvate, [RhCl(C(8)H(12))(C(24)H(23)N(2)O(4)P)].CH(2)Cl(2), has been determined at 173 K. The five-membered heterocycle of the phosphine ligand is in a slightly distorted twist conformation. An intramolecular N1-H1.Cl1 hydrogen bond contributes to the adopted conformation and may additionally participate in secondary interactions with substrates during catalysis.

A comparison of the ring conformational properties of two derivatives prepared from the same diene ­diacetate precursor

Results of single-crystal X-ray experiments performed for the title compounds, (1S,2R,3S,4R,5R)-4-benzyloxy-2-[1-(benzyloxy)allyl]-5-hydroxymethyl-2,3,4,5-tetrahydrofuran-3-ol, C(22)H(26)O(5), (I), and (3R,5S,6S,7S,8S)-3,6-bis(benzyloxy)-5-iodomethyl-2,3,4,5-tetrahydrofuro[3,2-b]furan-2-one, C(21)H(21)IO(5), (II), demonstrate that the tetrahydrofuran ring that is common to both structures adopts a different conformation in each molecule. Structural analyses of (I) and (II), which were prepared from the same precursor, indicate that their different conformations are caused by hydrogen-bonding interactions in the case of (I) and the presence of a fused bicyclic ring system in the case of (II). Density functional theory calculations on simplified analogs of (I) and (II) are also presented.

The lack of C2 molecular symmetry in (1R,2R,3S,6S)-3,6-dibenzyloxycyclohex-4-ene-1,2-diol.

The results of a single-crystal X-ray experiment and density functional theory calculations performed for the title compound, C20H22O4, demonstrate that the lowest energy conformation of this molecule does not contain C2 molecular symmetry.