Heme-Based Sensors, Exemplified by the Kinase FixL, Are a New Class of Heme Protein with Distinctive Ligand Binding and Autoxidation

Abstract

FixL's are chimeric heme protein kinases from symbiotic nitrogen-fixing Rhizobia. We have overexpressed three FixL variants in Escherichia coli. Bradyrhizobium japonicum FixL, a soluble dimeric protein, is the first full-length FixL to be purified. The other two proteins are soluble truncations of Rhizobium meliloti FixL, which is a membrane protein. One contains both heme and kinase domains and is dimeric; the other has only the heme domain and is monomeric. We find that all the FixL's bind oxygen and carbon monoxide non-cooperatively, with very low affinities due entirely to slow association rates. FixL P50's for oxygen are 17-76 mmHg. FixL's may sense nitric oxide and carbon monoxide in addition to oxygen, especially at the low oxygen pressures encountered in vivo. Autoxidation rates are about 50 times faster than that of sperm whale myoglobin. The carbon monoxide affinity of FixL's is about 300 times lower than that of myoglobin, resulting in the unusually low values of 7.5-17 for the partition constant, M = P50(O2)/P50(CO), between carbon monoxide and oxygen. Met-FixL's have their Soret absorption maximum at 395 nm instead of the typical 408 nm and a steep hydroxymet transition at pH > or = 9.3; these properties indicate a pentacoordinated high-spin ferric heme and suggest a sterically hindered hydrophobic heme pocket lacking a distal (E7) histidine. FixL is the first member of a new class of heme proteins, the heme-based sensors, distinct from the oxygen carriers and electron transporters. We expect that some of the novel properties of FixL will be characteristic of the class.

Mitochondrial Respiration and Hemoglobin Gene Expression in Barley Aleurone Tissue

“…There are several possible roles for Hb under hypoxic conditions: (a) it may serve as an O, carrier, like myoglobin (Wittenberg and Wittenberg, 1990), to allow mitochondrial respiration to continue during early anoxia; (b) it may act as an electron-transfer protein, as has been suggested for a flavohemoglobin from Esckerickia coli (Cooper et al, 1994) and the Vitreoscilla Hb (Chen et al, 1994;Kallio et al, 1994); and (c) it may function as a sensor to regulate expression of other genes, as has been shown for the heme protein FixL of Xkizobium meliloti (Gilles-Gonzalez and Gonzalez, 1993;Gilles-Gonzalez et al, 1994).…”

A Distal Arginine in Oxygen-Sensing Heme-PAS Domains Is Essential to Ligand Binding, Signal Transduction, and Structure

“…Interestingly, even with this guanido group present, the FixL dissociation equilibrium constants for binding of O 2 (K d ∼ 50-140 µM) are more than 50-fold higher than that of A. limacina myglobin (1,11). Instead, the affinities of FixL proteins fall within the range measured for hemes in entirely apolar media, such as free heme in benzene (12).…”

“…This latter "autophosphorylation" reaction is equally robust in deoxyand met-FixL but is strongly inhibited by binding of O 2 to the ferrous form or CN -to the ferric form (15,18). Although the B. japonicum and Sinorhizobium meliloti FixL/FixJ regulatory systems share these fundamental features, they differ in other important respects, including their affinities for heme ligands and precise regulatory targets (1,(19)(20)(21). The enzymatic regulation has been studied most thoroughly for the S. meliloti FixL kinase, RmFixLT, whereas the ligandinduced structural changes of the heme pocket have been observed only for the B. japonicum FixL heme-binding domain, BjFixLH (9,13).…”

Behavior of Rhizobium meliloti in oxygen gradients

“…Oxygen consumption by the respiring cells eratively and with low affinity; consequently, it can respond was measured using a Clark-type electrode. For the temporal assay of linearly to an extended range of oxygen concentrations [10].…”