This displacements probably induces a movement of helix A, which is believed to lead to disruption of the dimer interface, consisting of helices A and B. Tyr16 blocks the distal coordination site and has to be displaced to allow ligand binding to the heme. In cyt- c′, the vacant site is only accessible to the solvent via a narrow channel in the protein. Like in many gas-sensing proteins, the selectivity in ligand binding arises from steric hindrance of the vacant coordination site.
Cytochrome c′ from the purple photosynthetic bacterium Allochromatium vinosum (cyt- c′) displays a unique dimer-to-monomer transition upon binding of NO, CO, CN − or small alkyl isocyanides.
Most cytochromes c′ are isolated as stable homodimers, although some occur as monomers or as a mixture of both. Its CO and NO binding properties have been applied in electrochemical and fiberoptic fluorescent biosensors. Scientific interest in cytochromes c′ has increased further owing to the similarities in spectral properties and ligand binding properties between cytochrome c′ from Alcaligenes xylosoxidans and sGC. Although a role in electron transfer processes had been assumed for a long time, more recently it has been suggested that cytochromes c′ are involved in protection against high levels of NO. Despite their ubiquity in bacteria, the biological role of cytochromes c′ remains unclear. Similar to sGC, cytochromes c′ can bind small ligands like NO, CO and CN −, but not O 2. Unlike the six-coordinated iron in other cytochromes, the heme iron in cytochromes c′ is five-coordinate with a vacant distal coordination site. They consist of a four-helix bundle with a heme group covalently attached to a CKXCH motif near the C-terminus. The cytochromes c′ are a widespread class of cytochromes found in the periplasm of photosynthetic, denitrifying, nitrogen-fixing and sulfur-oxidizing bacteria and show many similarities to heme-based gas-sensor proteins. Via this pathway, NO is involved in various physiological processes such as immune response, vasodilation and neurotransmission. The generation of the second messenger 3′-5′-cyclic guanosine monophosphate by soluble guanylate cyclase (sGC) in mammals is regulated by binding of NO or CO. CO binding to the heme of the transcription factor CooA activates its DNA binding domain, initiating the transcription of enzymes that allow the photosynthetic bacterium Rhodospirillum rubrum to grow on CO as a sole energy source. Binding of O 2 to the regulatory heme-PAS domain of FixL is involved in the regulation of gene expression in nitrogen-fixing bacteria by inactivating the FixL kinase domain. The binding of diatomic gases such as O 2, CO or NO to gas-sensing heme proteins illustrates that even the binding of small molecules can cause large rearrangements in a protein’s secondary, tertiary or quaternary structure. Ligand-induced conformational changes of proteins are crucial for the regulation of protein activity in many biological processes. Comparison of ligand binding kinetics as observed with UV–vis spectroscopy with changes in fluorescence suggested that binding of one CO molecule per dimer could be sufficient for monomerization. Ligand binding to the heme and the dissociation of the dimer in solution were also studied using energy transfer from a fluorescent probe to both heme groups of the protein. The kinetics of the ligand-induced monomerization were found to be significantly enhanced in the gas phase compared with the kinetics in solution, however. Native mass spectrometry allowed the detection of the dimeric and monomeric forms of cytochrome c′ and even showed the presence of a CO-bound monomer. Here we have explored two biophysical techniques to simultaneously monitor ligand binding and monomerization. While ligand binding to the heme has been well characterized using a variety of spectroscopic techniques, direct monitoring of the subsequent monomerization has not been reported previously. This homodimeric protein dissociates into monomers upon binding of NO, CO or CN − to the iron of its covalently attached heme group. Cytochrome c′ from Allochromatium vinosum is an attractive model protein to study ligand-induced conformational changes.
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