ddhGTP The absence of direct oxidation of Fe by
The absence of direct oxidation of Fe2+ by L1 in experiments in solution would be consistent with the recognized low O2 affinity of sGC. It is known that, in ddhGTP with other hemoproteins, such as globins and heme-containing oxygenases with a Fe2+-proximal histidine linkage, sGC prevents oxidation of ferrous heme by O2 , , , . Fig. 6 compares the region of the I-type signals in cyclic voltammograms of sGC films in contact with aqueous phosphate buffer in the absence and in the presence of L1 at different potential scan rates. Peak splitting (I′ and I″ signals) can be seen in both cathodic and anodic waves when the potential scan is initiated at values positive enough to promote the initial Fe2+ to Fe3+ oxidation. Upon increasing the potential scan rate, the peak I′ is enhanced at the expense of the peak I″ which is considerably lowered in the presence of L1. These features can be interpreted on assuming that film formation implies some structural constraints resulting in the adoption of two different coordinative environments for the group heme, possibly derived from changes in the tertiary structure of the protein (absent in GC solutions where no peak splitting appeared, see Fig. 3a). The coordinative arrangement responsible for the signals at less positive potentials would be that more ‘open’, thus prompting a major ability to bound the heme unit with external species, as denoted by the increase of the signal I′ relative to the signal I″ in the presence of L1 and on decreasing the experimentation time (i.e. increasing potential scan rate) (see Fig. 7). Based on kinetic and mutational analyses, it is believed that the critical factor that may contribute to the low O2 affinity is the protein proximal effect, a regulatory effect caused by the weak bond between the Fe2+ and the proximal His , . Based on quantum mechanical and molecular mechanical analyses, it was proposed that the degree of electron density on the heme-iron might be controlled by the protein proximal effect, which in turn would modulate the Fe2+-ligand bond formation on the distal side . However, Marletta et al. have shown that the lack of O2 affinity by sGC is due to the lack of H-bonding amino acids in the active site of sGC . Comparison of electrochemical data in sGC plus L1 solutions and those at sGC and L1 solid films on glassy carbon electrodes suggests that, if structural/conformational constraints imposed by the solid sate in L1 and particularly, in sGC films are introduced, there is possibility of weakening the Fe2+-proximal histidine linkage thus enhancing promptly the oxidation of Fe2+ by L1. The observed splitting in peak I can be associated to the proximal effect derived by strain on the Fe2+-His bond probably affects redox potential of the heme, because the decrease in electron density at the ferrous heme makes it more difficult to remove an electron, as illustrated by the fact that the T-state hemoglobin with a more strained Fe2+-His bond and lower O2 affinity relative to R-state hemoglobin exhibited significantly higher midpeak potentials of the heme than R-state hemoglobin . However, the low magnitude of the peak potential shift suggests that L1 possibly does not affect the Fe-His bond (i.e., there is no direct binding to the heme) but binds to the sGC active site in different orientations. Both interpretations would require that L1 acts as a coordinating agent rather than a redox one. On the other hand, recent studies indicate that heme-bound NO only partially activates sGC and additional NO is involved in the mechanism of maximal NO activation , , . Then it was proposed that super-activation of sGC occurs via a “second site”. Fernhoff et al.  suggested that cysteine(s) residue(s) are involved in this second site activation. Accordingly, cystein(es) mediates NO activation providing a second NO binding site that produces sGC state (cys-NO) which would be more active than the ‘ordinary’ (termed 1-NO) state involving NO binding to Fe2+-heme. The rapid dissociation of the cysteine/NO complex deactivates the enzyme to the 1-NO state, which persists until NO slowly dissociates from the sGC heme and the enzyme reverts to the basal state . Recent data indicate that sGC undergoes a reductive nitrosylation reaction that is coupled to the S-nitrosation of sGC cysteines gated by a conformational change of the protein causing the NO desensitization of the ferric sGC .