Controlling Ligand Binding in Myoglobin by Mutagenesis

The rate of each transition was increased relative to wild-type bsNOS, with Fe III NO dissociation being 3.6 times faster. In V346I iNOSoxy we consecutively observed the beginning ferrous, Fe II O 2 , a mixture of Fe III NO and ferric heme species, and ending ferric enzyme. The rate of each transition was decreased relative to wild-type iNOSoxy, with the Fe III NO dissociation being 3 times slower. An independent measure of NO binding kinetics confirmed that V346I iNOSoxy has slower NO binding and dissociation than wild-type. Citrulline production by both mutants was only slightly lower than wild-type enzymes, indicating good coupling. Our data suggest that a greater shielding of the heme pocket caused by the Val/Ile switch slows down NO synthesis and NO release in NOS, and thus identifies a structural basis for regulating these kinetic variables.Nitric oxide synthases (NOSs) 1 are flavoheme enzymes that generate nitric oxide (NO) from L-arginine (Arg) (1, 2). The overall reaction consumes 1.5 NADPH and 2 O 2 and involves two steps, the first being Arg hydroxylation to form N-hydroxy-L-Arg (NOHA), and the second being NOHA oxidation to form citrulline and NO (see Scheme 1).The NOS heme is located in a catalytic "oxygenase" domain that also contains the binding sites for Arg and the essential redox cofactor (6R)-tetrahydrobiopterin (H 4 B) (3, 4). The heme is ligated to a cysteine thiolate (5-7) and catalyzes a reductive activation of molecular oxygen in conjunction with H 4 B in each of the two steps of NO synthesis (8 -11). The NOS heme also binds self-generated NO as an intrinsic feature of catalysis (12)(13)(14). Each molecule of NO generated by NOS has a high probability of binding to the heme before it is released from the enzyme. The resulting ferric heme-NO product complex (Fe III NO) has been observed to build up as a transient species during NOHA oxidation reactions catalyzed by NOS oxygenase domains (NOSoxy) when run in a stopped-flow spectrophotometer under single turnover conditions (9,13,14). These experiments also provided spectral and kinetic information regarding the formation of an initial ferrous heme-dioxy species (Fe II O 2 ), whose disappearance then coincides with k cat and formation of the transient Fe III NO product complex (see Scheme 2). Because dissociation of the Fe III NO product complex is required for NO to escape from the enzyme, it is an essential step for the biologic functions of animal NOSs. In fact, this dissociation rate is one of three key kinetic parameters that together determine the overall catalytic behavior of a given NOS (15,16). Slower rates of NO dissociation dispose the Fe III NO product complex toward its reduction during catalysis, which then places the ferrous heme-NO enzyme species into a futile cycle that does not release NO (Fig. 1). Interestingly, there is only a modest variation in Fe III NO dissociation rates among the three mammalian NOS isoforms, which range from 2 to 5 s Ϫ1 when measured in NOHA single turnover reactions at 10°C (16).Given the above, we...

Section: Discussionmentioning

confidence: 99%

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

Wang

Wei

Sharma

et al. 2004

“…Using mutagenesis, Wilkinson, Phillips, Olson, and their colleagues have demonstrated unambiguously that increasing the strength and number of hydrogen bonds donated from the distal pocket amino acids to bound O 2 lowers the rate of oxygen dissociation from Mb (39,41,55,56). The best example is V68N pig Mb, in which Val at the E11 helical position is replaced with Asn.…”

Section: Resultsmentioning

confidence: 99%

“…Association and dissociation rate constants for O 2 binding to the ferrous form of the heme-HmuO complex are compared with those of rat HO-1 and wild-type and V68N pig Mb in Table II (41). Errors for HO-1 were obtained from three different independent experimental determinations (different samples and different days) and indicate that there are no significant differences between the ligand binding parameters for HmuO and HO-1.…”

Section: Resultsmentioning

confidence: 99%

Crystal Structure of the Dioxygen-bound Heme Oxygenase from Corynebacterium diphtheriae

Unno

Matsui

Chu

et al. 2004

100

171

HmuO, a heme oxygenase of Corynebacterium diphtheriae, catalyzes degradation of heme using the same mechanism as the mammalian enzyme. The oxy form of HmuO, the precursor of the catalytically active ferric hydroperoxo species, has been characterized by ligand binding kinetics, resonance Raman spectroscopy, and x-ray crystallography. The oxygen association and dissociation rate constants are 5 M ؊1 s ؊1 and 0.22 s ؊1 , respectively, yielding an O 2 affinity of 21 M ؊1 , which is ϳ20 times greater than that of mammalian myoglobins. However, the affinity of HmuO for CO is only 3-4-fold greater than that for mammalian myoglobins, implying the presence of strong hydrogen bonding interactions in the distal pocket of

“…However, CerHb contains a Thr at the E11 position, and the Thr-48(E11) ␤-hydroxyl O-␥1 atom pulls the Tyr-11(B10) phenol hydrogen atom away from the bound ligand. As a result, the nonbonding electrons on the Tyr(B10) O-atom are pointing toward the bound ligand, and this negative partial charge causes the oxygen affinity of WT CerHb to be similar to that of vertebrate Mbs (K O2 Ϸ 1 M Ϫ1 ) (31,32). CerHb also displays very rapid rates of O 2 association (kЈ O2 ϭ 240 M Ϫ1 s Ϫ1 ) and dissociation (k O2 ϭ 180 s…”

mentioning

confidence: 99%

“…CerHb is considered to be in a unique mini-globin subcategory within the larger globin families (29) and has evolved a Mb-like O 2 storage function in the nerve system of the sea worm (30). Like many microbial and invertebrate globins, CerHb has a ligand-binding site containing Tyr(B10) and Gln(E7), which typically evokes an extremely high oxygen affinity because of formation of multiple hydrogen bonds with the bound ligand (28,31). However, CerHb contains a Thr at the E11 position, and the Thr-48(E11) ␤-hydroxyl O-␥1 atom pulls the Tyr-11(B10) phenol hydrogen atom away from the bound ligand.…”

mentioning

confidence: 99%

The Apolar Channel in Cerebratulus lacteus Hemoglobin Is the Route for O2 Entry and Exit

Salter

Nienhaus

et al. 2008

Self Cite

The major pathway for O 2 binding to mammalian myoglobins (Mb) and hemoglobins (Hb) involves transient upward movement of the distal histidine (His-64(E7)), allowing ligand capture in the distal pocket. The mini-globin from Cerebratulus lacteus (CerHb) appears to have an alternative pathway between the E and H helices that is made accessible by loss of the N-terminal A helix. To test this pathway, we examined the effects of changing the size of the E7 gate and closing the end of the apolar channel in CerHb by site-directed mutagenesis. Increasing the size of Gln-44(E7) from Ala to Trp causes variation of association (k O2 ) and dissociation (k O2 ) rate coefficients, but the changes are not systematic. More significantly, the fractions (F gem ≈ 0.05-0.19) and rates (k gem ≈ 50 -100 s ؊1) of geminate CO recombination in the Gln-44(E7) mutants are all similar. In contrast, blocking the entrance to the apolar channel by increasing the size of Ala-55(E18) to Phe and Trp causes the following: 1) both k O2 and k O2 to decrease roughly 4-fold; 2) F gem for CO to increase from ϳ0.05 to 0.45; and 3) k gem to decrease from ϳ80 to ϳ9 s ؊1, as ligands become trapped in the channel. Crystal structures and low temperature Fourier-transform infrared spectra of Phe-55 and Trp-55 CerHb confirm that the aromatic side chains block the channel entrance, with little effect on the distal pocket. These results provide unambiguous experimental proof that diatomic ligands can enter and exit a globin through an interior channel in preference to the more direct E7 pathway.The structural mechanisms for O 2 binding to myoglobins (Mb) 3 and hemoglobins (Hb) have been the topic of much research since Kendrew (1) and Perutz et al. (2) first reported the three-dimensional structures of Mb and Hb over 45 years ago. In the case of mammalian myoglobins, ligand binding is well understood (Refs. 3-10 and references therein) and consists of four major steps. Weakly bound water is displaced to create a vacant distal pocket above the heme iron atom. Ligands migrate into the protein through a short channel that is created when the distal histidine (His-64 at the E7 helical position) 4 transiently rotates upward and then are captured in the interior of the distal pocket. This noncovalent intermediate is often called the B state because it can also be generated by photolysis of the equilibrium bound or A state. Covalent bond formation between the internal ligand and the iron atom of the heme group then competes with ligand escape back out through the His(E7) gate. The bound ligand can be further stabilized by electrostatic interactions with surrounding polar amino acid side chains. This E7 gate pathway appears to occur in most if not all animal hemoglobins, with the classic 3-on-3 ␣-helical globin fold and a distal histidine, although experimental verification has only been done rigorously for vertebrate Mbs using time-resolved crystallography (4 -8), site-directed mutagenesis (3, 9, 11), and time-resolved absorbance and FTIR spectroscopy (12-18).In con...