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COMPARISON OF THE RATES OF NITRIC OXIDE GEMINATE RECOMBINATION TO THE HEME OF eNOS AND OF nNOS

The above differences between eNOS and nNOS in terms of NO release and heme nitrosy-lation are related to the stability of their ferrous nitrosyl complexes. For nNOS, this ferrous nitrosyl complex of nNOS is very stable. The rates for spontaneous binding and dissociation of NO to nNOS were reported as kon = 2 x 107 M-1s-1 and koff = 1 x 10-4s-1 [60]. This is consistent with the majority of the nNOS protein that exist as a ferrous heme-NO complex in steady-state (aerobic) catalysis, since the nitrosylation governs and limits the rate of NO synthesis [61]. In contrast, in eNOS, little heme-NO complex forms during normal turnover when arginine is the substrate. The nitrosylation rate of ferrous eNOS was reported as kon = 1.1 x 106 M-1s-1 whereas conflicting values were published for the dissociation rate: koff = 70 s-1 and 6 x 10-4 s-1 [23,54]. These dissociation rates are at least an order of magnitude larger than for nNOS. These numbers explain the experimental fact that eNOS has a significantly lower degree of nitrosylation than nNOS, once NO is formed via bimolecular recombination of NO to the proteins. The kinetics of photo-induced release and rebinding of free NO from the ferrous nitrosylated eNOS and nNOS were measured by ultra-fast absorption spectroscopy (geminate rebinding of the nitrosyl ligand after photodissociation). The buffered solution had a high 50 ^M concentration of ferrous protein that was nitrosylated by prolonged bubbling of the solution with argon containing 0.1% NO. Optical absorption confirmed that bubbling achieved full nitrosylation of the heme within the experimental error of ca 5%. We used a 30 fs pump pulse of 565 nm to selectively cleave the NO-heme bond of the ferrous nitrosyl complex. This bond cleavage generated the unliganded Fe2+ heme at the expense of the ferrous nitrosyl complex. The kinetics of the absorption spectrum after bond cleavage was monitored with a wideband probe beam (between 390 and 550 nm) with high time resolution. A typical trace of the kinetics is shown in Fig. 8. The amplitude AOD represents the change in optical density by application of the pump pulse. At given optical pathlength d, this observable value is proportional to the concentration of non-nitrosyl heme [Fe2+] released by the pump pulse

AOD (t = 0) = d(£410(heme) - £410(NO—heme)} • [Fe2+]

Significantly, the optical density does not return to its original value just prior to the pump pulse, but it approaches a positive asymptotic value. This phenomenon is particularly manifest for the eNOS isoform, but applies to nNOS as well. It proves that not all cleaved NO-heme bonds are restored by recapture of the nitrosyl ligand. Phrased otherwise, a certain fraction of NO ligands was actually released from the enzyme as free NO.

Fig. 8. Kinetics of the optical density at 410 nm due to geminate recombination of NO ligands to the ferrous heme of eNOS and nNOS. The reaction is initiated by a short pump pulse at 565 nm. The solutions contain ca 50 ^M nitrosylated eNOS after bubbling with argon containing 0.1% NO. The kinetic constants are given in Table 4. The decay curves are fit according to Eq. (4).

Fig. 8. Kinetics of the optical density at 410 nm due to geminate recombination of NO ligands to the ferrous heme of eNOS and nNOS. The reaction is initiated by a short pump pulse at 565 nm. The solutions contain ca 50 ^M nitrosylated eNOS after bubbling with argon containing 0.1% NO. The kinetic constants are given in Table 4. The decay curves are fit according to Eq. (4).

Table 4 Kinetic constants of the geminate recombination of NO ligands to ferrous heme of eNOS and nNOS. The parameter A4 represents the probability that the photodissociated NO is released from the protein and escapes recombination with the heme eNOS

nNOS

As often found for the recombination kinetics of hemeproteins, NO rebinding kinetics could be described as the sum of three exponentials with clearly distinct timescales ti < T2 < T3 where A1 + A2 + A3 + A4 = 1

The first exponential (A1, T1) accounts for rapid NO rebinding from the immediate vicinity to the heme iron, and is activationless. The second and third exponentials account for much slower kinetics where the NO rebinding takes place from larger distances to the heme, but still within the heme pocket or its vicinity. These phases are usually required to overcome internal energy barriers for NO recombination to the heme. The constant term A4 is the probability of escape of free NO from the protein. It is clearly seen from Fig. 8 that this escape probability A4 is much higher in eNOS than in nNOS (0.20 versus 0.06). It confirms that very little NO is able to escape from the reduced nNOS. This result is in complete agreement with the absence of spontaneous (thermolytic) release of NO from nitrosylated nNOS and the high degree of nitrosylation of nNOS in the presence of nitrite (cf Fig. 7). This NO-bound ferrous complex probably arises from NO generated by nitrite reduction, but is unable to escape from the ferrous nNOS. In contrast, once oxygen is able to oxidize the heme to its ferric state, this NO should be released immediately in a "burst." This NO burst could be very toxic since it happens as the oxygen is freshly supplied, for example, at reperfusion. Moreover, it can combine with the superoxide ions formed under decoupling conditions to generate peroxynitrite. This could be the basis of the reported evidence of nNOS promoting inflammation in the cerebral microcirculation whereas eNOS blunts the extent of this response to episodic hypoxia and exerts neuroprotective effects [62].

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