Electron Transfer, Oxygen Binding, and Nitric Oxide Feedback Inhibition in Endothelial Nitric-oxide Synthase

Abstract

We studied steps that make up the initial and steadystate phases of nitric oxide (NO) synthesis to understand how activity of bovine endothelial NO synthase (eNOS) is regulated. Stopped-flow analysis of NADPH-dependent flavin reduction showed the rate increased from 0.13 to 86 s ؊1 upon calmodulin binding, but this supported slow heme reduction in the presence of either Arg or N -hydroxy-L-arginine (0.005 and 0.014 s ؊1 , respectively, at 10°C). O 2 binding to ferrous eNOS generated a transient ferrous dioxy species (Soret peak at 427 nm) whose formation and decay kinetics indicate it can participate in NO synthesis. The kinetics of heme-NO complex formation were characterized under anaerobic conditions and during the initial phase of NO synthesis. During catalysis heme-NO complex formation required buildup of relatively high solution NO concentrations (>50 nM), which were easily achieved with N -hydroxy-L-arginine but not with Arg as substrate. Heme-NO complex formation caused eNOS NADPH oxidation and citrulline synthesis to decrease 3-fold and the apparent K m for O 2 to increase 6-fold. Our main conclusions are: 1) The slow steady-state rate of NO synthesis by eNOS is primarily because of slow electron transfer from its reductase domain to the heme, rather than heme-NO complex formation or other aspects of catalysis. 2) eNOS forms relatively little heme-NO complex during NO synthesis from Arg, implying NO feedback inhibition has a minimal role. These properties distinguish eNOS from the other NOS isoforms and provide a foundation to better understand its role in physiology and pathology. Nitric-oxide synthases (NOSs)1 catalyze a stepwise oxidation of L-arginine (Arg) to citrulline and nitric oxide (NO) (1-3). In mammals, three NOSs are expressed that differ in their primary sequence, post-translational modifications, cellular location, and tissue expression (4 -6), consistent with their participating in a range of physiologic and pathologic systems. Two NOSs (neuronal, nNOS or NOS-I; and endothelial, eNOS or NOS-III) are constitutively expressed and participate in signal cascades by synthesizing NO in response to Ca 2ϩ -dependent CaM binding. A third NOS (cytokine-inducible, iNOS or NOS-II) is primarily regulated by transcriptional mechanisms, binds CaM irrespective of the Ca 2ϩ concentration to be always active, and functions as both a regulator and effector of the immune response.Although NO synthesis activities of the NOS isoforms differ considerably, how and why this occurs is unclear. A comparison of published steady-state rates shows that eNOS is about four to eight times slower than either nNOS or iNOS (7-14). Because NO synthesis is actually the result of many steps, it is imperative to identify which steps limit the activity of a particular NOS isoform. Work with NOS chimeras containing swapped reductase domains has suggested heme reduction could be responsible for the low activity of eNOS (30). However, it seems that NOS catalysis is comprised of two parts (15, 16): an active component that includes...

A Conserved Val to Ile Switch near the Heme Pocket of Animal and Bacterial Nitric-oxide Synthases Helps Determine Their Distinct Catalytic Profiles

“…At this point, we can assume that all of the kinetic effects are because of the mutations either increasing or decreasing heme pocket access. Thus, one can rationalize that Ile supports slower rates of Fe II O 2 formation and extrinsic NO binding, because O 2 and NO access to the heme are similarly influenced by protein structural features in NOS (22,27,28) and in other hemeproteins (29). In contrast, the mutational effects on k cat were unexpected and cannot be so easily explained.…”

Differences in Three Kinetic Parameters Underpin the Unique Catalytic Profiles of Nitric-oxide Synthases I, II, and III

“…Protein Expression and Purification-Wild-type iNOS (full-length and oxygenase domains) and eNOS (full-length) proteins were expressed in Escherichia coli and purified as previously described (31,(33)(34)(35). UV-visible spectra were recorded on an Hitachi U3110 spectrophotometer in the absence or presence of 20 M H 4 B and 1 mM Arg.…”

“…Otherwise, a k cat of 36 s Ϫ1 was determined at 10°C using NOHA as a substrate in singleturnover experiments. 2 The rate of iNOS ferric heme reduction (0. binding to ferrous iNOS was from Abu-Soud et al (31). Computer simulations were run as described previously for nNOS (18; see "Experimental Procedures").…”

“…Regarding eNOS, very little heme⅐NO complex forms during normal turnover and is associated with no detectable inhibition of catalysis (31). However, when NOHA was used in place of Arg as substrate to create a faster rate of NO synthesis, there was greater heme⅐NO complex formation and measurable catalytic inhibition, which was linked to NO accumulation in the medium.…”

High Dissociation Rate Constant of Ferrous-Dioxy Complex Linked to the Catalase-like Activity in Lactoperoxidase

“…Weakening the Fe-O 2 linkage might allow the ligand trans to O 2 to pull the iron out of the plane away from the O 2 . This course of action is associated with the generation of a new oxygenated LPO species that can be identified by its Soret absorbance peak centered at 416 nm (reaction 13) similar to oxyhemoglobin and nitric oxide synthases (46,(47)(48)(49). The later intermediate is unstable, and its formation and autooxidation to ground state, which involves electron transfer to O 2 as a primary step to form superoxide (reaction 14), occurs in an oxygen-independent fashion.…”