Determinants of ligand affinity and heme reactivity in H-NOX domains.
Determinants of ligand affinity and heme reactivity in H-NOX domains.
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DOI：
10.1002/anie.200904799
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发表时间：
2010
影响因子：
16.6
通讯作者：
Marletta, Michael A.
中科院分区：
文献类型：
--
作者：
Weinert, Emily E.;Plate, Lars;Whited, Charlotte A.;Olea, Charles, Jr.;Marletta, Michael A.
Investigations into the mechanisms by which heme proteins control ligand affinity and reactivity have been studied for decades, with the globins serving as model systems for histidyl-ligated proteins.[1] The discovery of a novel family of heme proteins, Heme Nitric oxide/OXygen (H-NOX) binding domains,[2–4] has provided an opportunity to investigate if factors previously found to control ligand affinity and reactivity in the globins can be generalized between protein folds or if there are additional determinants. The H-NOX family includes the heme domain of soluble guanylate cyclase (sGC), the mammalian NO receptor,[5] as well as heme proteins from bacteria, such as Thermoanaerobacter tengcongensis (Tt).[3] H-NOX domains are a potentially illuminating choice for further investigations into ligand affinity because sGC does not bind O2, even under ambient conditions, whereas other members of the family, such as Tt H-NOX, bind O2 with very high affinity (Kd= 90 nm).[3, 6] A distinctive feature of the wild-type (WT) Tt H-NOX crystal structure was a distal-pocket hydrogen-bonding network, in which a key H bond from Y140 to the bound O2 was observed, as well as H bonds from N74 and W9 to Y140 (Figure 1).[4] The absence of this hydrogen-bonding triad in H-NOX domains that do not bind O2 highlights hydrogen bonding as a major component of O2 binding. A distal H bond to O2 has been noted to be important for the globins. However, the introduction of a tyrosine residue into sGC does not result inO2 binding,[7] which suggests that there are additional determinants of ligand affinity in H-NOX domains. To further investigate how the H-NOX fold regulates ligand binding, and how this regulation is similar or dissimilar to that of the globins, site-directed mutagenesis was performed on Tt H-NOX. It has been found that the introduction of phenylalanine mutations into the distal pocket of myoglobin (Mb), at positions such as L29 and V68, dramatically alters the oxygen affinity by decreasing the size of off-heme ligand-binding sites and reducing ligand “trapping”(Table 1).[8–10] In addition to altering O2 affinity, reaction of ferrous-oxy proteins with NO, or NO dioxygenation (Scheme 1, Table 1), in Mb was decreased by the addition of distal pocket bulk due to the blocking off-heme binding sites, preventing NO from readily residing in the heme pocket near the bound O2.[8, 11] To probe the effects of distal-pocket bulk on the O2 affinity and reactivity toward NO of H-NOX proteins, as compared to the globins, isoleucine75 and leucine144 were mutated to phenylalanine, since these residues are both in the back of the distal pocket (Figure 1) in positions analogous to those of L29 and V68 in Mb. The I75F, L144F, and I75F/L144F mutants were spectrally similar to WT Tt H-NOX (see Figure S1 in the Supporting Information); however, kinetic characterization revealed dramatic changes upon the introduction of distal-pocket bulk. The I75F mutation resulted in an approximately ninefold increase in the O2 off-rate (koff= 11.19 sÀ1), whereas the L144F mutation resulted in an approximately 13-fold increase in the O2 off-rate (koff= 16.06 sÀ1; Table 1). The two mutations had a synergistic effect in the I75F/L144F double mutant, which had an O2 off-rate of 45.7 sÀ1. Thus, a 37-fold increase was observed in the O2 off-rate upon the introduction of only two mutations, neither of which involved a direct change in the hydrogen-bonding network. Typically, such large changes in O2 off-rates occur when the primary hydrogen-bond donor to O2 is altered or removed.[9] There were also significant changes in the O2 on-rate upon introduction of the mutations (Table 1). These changes in …
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影响因子：
4.4
作者：
Iyer LM;Anantharaman V;Aravind L
通讯作者：
Aravind L
影响因子：
2.9
作者：
Eich, RF;Li, TS;Olson, JS
通讯作者：
Olson, JS
DOI：
10.1073/pnas.98.2.479
发表时间：
2001-01-16
影响因子：
11.1
作者：
Das, TK;Couture, M;Rousseau, DL
通讯作者：
Rousseau, DL
影响因子：
2.9
作者：
Karow, DS;Pan, DH;Marletta, MA
通讯作者：
Marletta, MA
影响因子：
56.9
作者：
Nioche, P;Berka, V;Raman, CS
通讯作者：
Raman, CS