Our data suggest that either nitrosylation or trans-nitrosylation act as a “gear shift” for myosin, switching it on-the-fly from a relatively high-speed, low-force motor to a low-speed, high-force motor at physiological concentrations of NO. The question naturally arises, to what purpose? Regulation of contraction under physiological and pathological conditions by NO is complex, involving direct and cGMP-dependent pathways 
, so a simple answer is unlikely to suffice. It has been proposed that the direct effect of NO in muscle cells is to slow contraction and its associated metabolism 
. One possible example of this is exercise, during which NO increases in skeletal muscle 
. NO or S-nitrosothiols may thus serve both to increase oxygen supply to the tissue through vascular dilation and simultaneously shift contractile function to higher force generation at the expense of lowered shortening velocity.
The concentrations of S-nitrosothiols used in these experiments are thought to span the physiological range. Free [NO] on the order of 100 nM to several µM has been measured adjacent to stimulated cardiac myocytes 
. However, the presence of these levels of free radical in the presence of ~200 µM concentrations of myoglobin in the myoplasm seems unlikely 
. It is entirely possible that the concentrations of nitrosothiols in myocytes and other cells are ~100 nM 
. However, as with any reversible bimolecular reaction, it is not only the concentration of reactants but the forward and reverse reaction rate constants for the nitrosylation reaction that will ultimately determine the functional impact of NO production. The forward reaction we know from motility experiments occurs in seconds. We also know that the effects of NO donors persist for at least several minutes once donor is removed, presumably due to slow spontaneous reversal of the S-NO modification. Thus S-NO-myofibrillar proteins may accumulate in cells, even in the presence of myoglobin.
The pattern of effects on force and actin filament velocity suggests a model where S-NO modification of muscle myosin alters the attached time and duty cycle of myosin. From the perspective of a single molecule, the velocity of actin over a pure myosin is related to the inverse of its attached lifetime (ton
) – how long during each hydrolytic cycle myosin remains attached to actin. In contrast, the time-averaged force generated by myosin is related to its duty cycle (ton
)) – the fraction of the total cycle time myosin is attached to actin. Thus a doubling of ton
with no change in the detached time (toff
) would result in a 50% decrease in actin filament velocity, and an approximate 2X increase in average force. Striated muscle myosins are thought to have low duty cycles; thus doubling ton
could produce the observed changes in force and velocity without large changes in total cycle time as measured by ATPase rates. This may explain why Nogueira and coworkers 
found no effect of DEA NONOate on ATPase rates; in addition, in those particular experiments their readout of myosin function was a non-functional, ion-activated ATPase assay rather than a functional actin-activated assay.
One might speculate that nitrosylation is a general regulatory mechanism for myosin-based motility. There are a number of cysteines that are well conserved across myosin isoforms, including two especially reactive cysteines 
(cys707 and cys697) among the nine in the motor domain of the heavy chain. Interestingly, the reactive cysteines themselves and the encompassing alpha helix are highly conserved in muscle myosins, even across species. However, conservation of the reactive cysteines does not fully extend to non-muscle myosins. Myosin V, for example, possesses one of the two reactive cysteines and conserves much of the encompassing helix from muscle myosins while other myosins have neither reactive cysteine. If one or both reactive cysteines are indeed the point of NO regulation of muscle myosin, then one would predict that myosins lacking these cysteines would be unresponsive to NO and nitrosothiols. It is possible, however, that non-muscle myosins incorporate different sites for regulation by NO, including one proposed in myosin heavy chain 9 
, that better meet their particular regulatory needs. Further, we found that some of the light chains of striated myosins can be nitrosylated in vitro
. There are obviously several possible sites for regulation of myosins by NO and its endogenous donors. Determining which of these is responsible for the effects observed here will be the subject of future studies, as will the identification of other potential NO regulatory sites in the contractile apparatus of cells.