Cytochrome c' binds and transfers endogenous NO in the denitrifying variant of Rhodobacter sphaeroides2.4.3 and the NO bound ferrous heme that forms 5-coordinate resembles regulatory site in soluble guanylate cyclase. The Arg127 is close to the 5-coordiate NO bound heme of the cytochrome c'. The mutant (R127A) that was overexpressed in a cytochrome c' knock-out R. sphaeroides2.4.3 provided the opportunity to research the functional role of Arg127 in the redox potential and NO binding. The mutant (R127A) showed lower redox potential and lower NO binding free energy than wild type. ERP and ENDOR showed that the mutant had less value of ¹⁴NO hyperfine coupling, which was closer to that of a 5-coordinate NO-heme model. Deuterium hyperfine coupling of NO wild type that showed the possibility of the hydrogen bonding with the O of the NO, disappeared at the mutant (R127A) with uncharged Ala127. Therefore wild type cytochrome c' with Arg127 has a higher NO dissociation constant for the NO bound heme and keeps the internal NO physiological micromolar concentration in the cell. The wild type with Arg127 provides a higher redox potential to favor the ferrous heme form for NO binding.

The burst phase of the spin labeled iso-1-cytochromes c was directly probed using a renovated continuous-flow EPR method with ∼ 65 μs resolution time and ∼ 0.21 μL resolution volume. The spin labeled cytochromes c provided the opportunity to research a specific residue response during the burst phase. There was an apparently global collapse for the spin labeled cytochromes c during sub-millisecond due to denaturant dilution. At a given temperature, the collapse times and the activation energy barriers were similar from one label position to another due to buffer viscosity.