A conserved flavin-shielding residue regulates NO synthase electron transfer and nicotinamide coenzyme specificity

Abstract

Nitric oxide synthases (NOSs) are flavoheme enzymes that contain a ferredoxin:NADP(+)-reductase (FNR) module for binding NADPH and FAD and are unusual because their electron transfer reactions are controlled by the Ca(2+)-binding protein calmodulin. A conserved aromatic residue in the FNR module of NOS shields the isoalloxazine ring of FAD and is known to regulate NADPH binding affinity and specificity in related flavoproteins. We mutated Phe-1395 (F1395) in neuronal NOS to Tyr and Ser and tested their effects on nucleotide coenzyme specificity, catalytic activities, and electron transfer in the absence or presence of calmodulin. We found that the aromatic side chain of F1395 controls binding specificity with respect to NADH but does not greatly affect affinity for NADPH. Measures of flavin and heme reduction kinetics, ferricyanide and cytochrome c reduction, and NO synthesis established that the aromatic side chain of F1395 is required to repress electron transfer into and out of the flavins of neuronal NOS in the calmodulin-free state, and is also required for calmodulin to fully relieve this repression. We speculate that the phenyl side chain of F1395 is part of a conformational trigger mechanism that negatively or positively controls NOS electron transfer depending on the presence of calmodulin. Such use of the conserved aromatic residue broadens our understanding of flavoprotein structure and regulation.

Figure 1

Location of F1395 in nNOS. (Upper) Stacking interaction between phenyl of F1395 (green) and FAD isoalloxazine ring (blue) in a crystallized FNR fragment of nNOS (12). Left and Right of Upper are zoom out and 300% zooming picture of the FNR module of nNOS, respectively. (Lower) C-terminal sequence alignments for rat neuronal NOS (nNOS, SWISS-PROT accession no. P29476), mouse inducible NOS (iNOS, accession no. P29477), bovine endothelial NOS (eNOS, accession no. P29473), rat cytochrome P450 reductase (CYPR, accession no. P00388), spinach ferredoxin:NADP⁺ reductase (FNR, accession no. P00455), Escherichia coli. sulfite reductase flavoprotein α-component (SIR-FP, accession no. P38038), and Caenorhabditis elegans methionine synthase reductase (MSR, accession no. Q17574). The conserved aromatic residue is marked by red.

Figure 2

Kinetics of NADPH-dependent flavin and heme reduction in F1395Y and F1395S. Flavin reduction was followed at 485 nm under anaerobic conditions at 10°C after mixing excess NADPH with CaM-bound (Top) or CaM-free (Middle) F1395Y and F1395S proteins. (Bottom) Heme reduction was detected by CO binding under anaerobic conditions and the kinetics were determined from the change in absorbance at 444 nm with time. CaM-bound enzymes were rapidly mixed with excess NADPH to trigger flavin and heme reduction at 10°C. Data shown are an average of 7–10 individual scans.

Figure 3

Stability of reduced flavins. The reduction and reoxidation of reduced flavins was monitored for wild-type or mutant nNOS at 485 nm. nNOS proteins were diluted to 1.5 μM in air-saturated 40 mM EPPS buffer, pH 7.6, containing 0.9 mM EDTA, and reactions were started by adding 60 μM NADPH at room temperature.