Article pubs.acs.org/biochemistry
Role of Reversible Histidine Coordination in Hydroxylamine Reduction by Plant Hemoglobins (Phytoglobins) Navjot Singh Athwal, Jagannathan Alagurajan, Amy H. Andreotti, and Mark S. Hargrove* The Roy J. Carver Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, Iowa 50011, United States S Supporting Information *
ABSTRACT: Reduction of hydroxylamine to ammonium by phytoglobin, a plant hexacoordinate hemoglobin, is much faster than that of other hexacoordinate hemoglobins or pentacoordinate hemoglobins such as myoglobin, leghemoglobin, and red blood cell hemoglobin. The reason for differences in reactivity is not known but could be intermolecular electron transfer between protein molecules in support of the required two-electron reduction, hydroxylamine binding, or active site architecture favoring the reaction. Experiments were conducted with phytoglobins from rice, tomato, and soybean along with human neuroglobin and soybean leghemoglobin that reveal hydroxylamine binding as the rate-limiting step. For hexacoordinate hemoglobins, binding is limited by the dissociation rate constant for the distal histidine, while leghemoglobin is limited by an intrinsically low affinity for hydroxylamine. When the distal histidine is removed from rice phytoglobin, a hydroxylamine-bound intermediate is formed and the reaction rate is diminished, indicating that the distal histidine imidazole side chain is critical for the reaction, albeit not for electron transfer but rather for direct interaction with the substrate. Together, these results demonstrate that phytoglobins are superior at hydroxylamine reduction because they have distal histidine coordination affinity constants near 1, and facile rate constants for binding and dissociation of the histidine side chain. Hexacoordinate hemoglobins such as neuroglobin are limited by tighter histidine coordination that blocks hydroxylamine binding, and pentacoordinate hemoglobins have intrinsically lower hydroxylamine affinities.
H
reduction of nitrite and HA by phytoglobins are generally much faster than those for neuroglobin, cytoglobin, and the pentacoordinate oxygen transport hemoglobins found in animals.1,2,20,21 The reduction of HA by phytoglobins is interesting because it requires the delivery of two electrons to make the stable endproduct ammonium, yet each ferrous phytoglobin heme delivers only one electron (Scheme 1).21 We have hypothesized that one Phyt2+ molecule binds HA and then accepts an electron from another Phyt2+ molecule to form ammonium and two molecules of Phyt3+ (Scheme 2).
exacoordinate hemoglobins (hxHbs) are found in all plants and animals, and some bacteria. They are defined by a globin fold that surrounds a binding site for heme b with two sources of iron coordination (most often two histidine side chains). One histidine remains coordinated to the heme iron akin to the “proximal” histidine of blood cell hemoglobin, and the other binds reversibly, allowing for competitive binding of other molecules such as oxygen, carbon monoxide (CO), nitric oxide (NO), nitrite, hydroxylamine (HA), cyanide, water, or hydroxide, among others. Proposed functions for hexacoordinate hemoglobins in plants and animals include NO production,1,2 NO scavenging,3−7 oxygen transport,8−11 and cell signaling.2,12−16 Recent interest has focused on hypoxic nitrite and HA reduction by hxHbs, including the plant nonsymbiotic hemoglobins “phytoglobins” (Phyt)17 and neuroglobin in animals. It is thought that the NO produced from nitrite reduction might have a role in signaling18,19 and that nitrite and HA reduction might be related to hypoxic metabolism in plants.4 Regardless of function, the rates of © 2016 American Chemical Society
Scheme 1
Received: July 28, 2016 Revised: September 19, 2016 Published: September 23, 2016 5809
DOI: 10.1021/acs.biochem.6b00775 Biochemistry 2016, 55, 5809−5817
Article
Biochemistry
PhytH2+, ferrous hexacoordinate Phyt; PhytP2+, ferrous pentacoordinate Phyt; Phyt3+, ferric Phyt; k−his, rate constant for dissociation of histidine from PhytH2+; k+his, rate constant for association of histidine with PhytH2+; kHA, rate constant for association of HA with PhytH2+; k−HA, rate constant for dissociation of HA from PhytH2+.
that have different rate and equilibrium constants for hexacoordination. Our results demonstrate that the pentacoordinate phytoglobin species (PhytP) rapidly binds HA with high affinity, and that PhytH quickly donates the second electron to complete the reaction. Reaction of the remaining fraction of PhytH is limited by histidine dissociation (k−his) to allow HA binding. Thus, in hxHbs with Khis values of ≫1 and slower values of k−his (such as neuroglobin), the reaction is slow because there is little pentacoordinate protein, and the reaction with the hexacoordinate species is limited by the lower value of k−his. We also demonstrate that the distal histidine is critical for HA reduction reaction, and that pentacoordinate hemoglobins, while capable of rapid reaction rates, are slowed by lower affinities for HA.
Electron transfer between Phyt molecules is rapid enough to support the previously observed rates of HA reduction,22 but it is not yet understood why phytoglobins conduct the reaction so much faster than other hxHbs such as neuroglobin and cytoglobin, and the oxygen transport hemoglobins myoglobin and leghemoglobin. An important distinction between phytoglobins and other hemoglobins is the fraction of hexacoordinate and pentacoordinate states found at rapid equilibrium in their populations (Figure 1).23,24 The class 1 phytoglobins exist in solution with ∼50% of the molecules coordinated by the distal histidine, and ∼50% in the pentacoordinate state, in which the distal histidine side chain is present in the heme pocket but not coordinated to the heme iron. In rice phytoglobin, the equilibrium constant for distal histidine coordination (Khis) is 1.9, which indicates a 65/ 45 ratio of hexacoordinate (PhytH) to pentacoordinate (PhytP) species (Figure 1).23 These populations interconvert with rate constants of 40 and 75 s−1 for histidine dissociation (k−his) and rebinding (k+his), respectively. It is this mixture of states that reacts with ligands, and CO and oxygen binding show distinct phases for reactions with the hexacoordinate and pentacoordinate species.25 It is not yet known whether physical properties such as histidine coordination are responsible for the stark differences in HA reduction rates between plant phytoglobins and other hemoglobins. In our experiments, we have used kinetic methods to study reduction of HA by rice phytoglobin, rice phytoglobin mutant proteins, and natural hemoglobin variants
EXPERIMENTAL PROCEDURES Preparation of Proteins. All proteins were expressed in Escherichia coli BL21 (DE3) cells (Agilent catalog no. 200131) and purified as reported previously.21,26,27 Briefly, the host cells were transformed with pET28 plasmids containing cDNA encoding the respective protein. The transformed E. coli cells were grown overnight at 37 °C while being shaken at 200 rpm in 2 L flasks containing 1 L of terrific broth medium. The harvested cells were concentrated and lysed by sonication, and the protein was purified using His tag affinity chromatography. Hydroxylamine Reduction Reactions. All reactions were conducted inside an anaerobic chamber maintained at 8% hydrogen in argon (Coy laboratories). Anaerobic 100 mM K2HPO4 buffer (pH 7.0) was prepared by boiling and subsequently flushing with nitrogen for 30 min. Deoxy-ferrous proteins were prepared using sodium dithionite followed by desalting over a Sephadex G-25 column to remove excess dithionite. The reactions were set up as described previously.21 A Bio-Logic stopped flow reactor (SFM-400, MOS-200) housed in the anaerobic chamber was used to collect kinetic traces in the visible region (555 nm for rice phytoglobin) using each heme protein at ∼20 μM in a flow cuvette with a 1 cm path length. Biokine software was used for data collection, followed by processing and fitting in Igor Pro. Numerical integration of Scheme 2 for Figure 2B used Dynafit.28 Anaerobic hydroxylamine (HA) was prepared by dissolving a preweighed amount of hydroxylamine hydrochloride salt in 100 mM K2HPO4 (pH 7.0) anaerobic buffer. Error bars shown in
Scheme 2. Species and Rate Constants Involved in the Reaction of HA with Phyta
a
■
Figure 1. Hexacoordination and hydroxylamine reduction by rice phytoglobin. The distal histidine side chain binds and dissociates from the heme iron with rate constants k+his and k−his, respectively, resulting in equilibrium constant Khis. Hydroxylamine binds the PhytP2+ conformation, which then reacts with another PhytH2+, resulting in the oxidation of both Phyt molecules and the associated two-electron reduction of hydroxylamine to form ammonium. 5810
DOI: 10.1021/acs.biochem.6b00775 Biochemistry 2016, 55, 5809−5817
Article
Biochemistry
CO concentration (Figure 4C) obey eq 3 and exhibit the asymptote representing the dissociation rate constant for HA.
■
RESULTS Kinetic Analysis of the Reaction Mechanism. The hypothesized mechanism for reduction of HA by phytoglobin is shown in Scheme 2 and Figure 1, along with rate and equilibrium constants for hexacoordination that are specific to rice Phyt. In this mechanism, HA reacts with the equilibrium mixture of PhytH and PhytP to form the Phyt−HA complex, which then reacts with another ferrous PhytH for electron transfer and product formation. Earlier work21 observed a linear relationship between HA reduction rate constants and HA concentration at lower concentrations but did not measure the rate constant at the higher concentrations needed to test this reaction mechanism. The experiments leading to the results depicted in Figure 2 were designed to test the kinetic limit of the reaction. The HA concentration was varied between 0 and 50 mM, and reaction time courses were measured as the change in absorbance at 558 nm associated with Phyt2+ oxidation. As previously reported, the spectra undergo the isosbestic transition from those of ferrous Phyt2+ to ferric Phyt3+ with no evidence of a stable intermediate.21 It is evident from these time courses (Figure 2A) that at even the lowest HA concentration there is a significant loss of absorbance amplitude in the reaction dead time (3 ms). This is expected if kHA and kET are much faster than k−his and k+his,25,27 as HA binding directly samples the fraction of PhytP in the starting reaction mixture. In fact, Scheme 2 predicts that under these conditions the fraction of amplitude lost in the dead time should be twice the fraction of Phytp because reaction 3 consumes a second molecule of Phyt2+. For rice Phyt, the fraction of PhytP is 35%.25 The total absorbance amplitude of this reaction is 0.23 absorbance unit, and the amount of amplitude lost at the highest HA concentration is 0.15, reflecting 65% of the reaction and nearly twice the fraction of PhytP. The data in Figure 2A thus suggest that PhytP binds HA in
