Plants express three phylogenetic classes of hemoglobins (Hb) based on sequence analyses. Class 1 and 2 Hbs are full-length globins with the classical eight helix Mb-like fold, whereas Class 3 plant Hbs resemble the truncated globins found in bacteria. With the exception of the specialized leghemoglobins, the physiological functions of these plant hemoglobins remain unknown. We have reviewed and, in some cases, measured new oxygen binding properties of a large number of Class 1 and 2 plant nonsymbiotic Hbs and leghemoglobins. We found that sequence classification correlates with distinct extents of hexacoordination with the distal histidine and markedly different overall oxygen affinities and association and dissociation rate constants. These results suggest strong selective pressure for the evolution of distinct physiological functions. The leghemoglobins evolved from the Class 2 globins and show no hexacoordination, very high rates of O(2) binding ( approximately 250 muM(-1) s(-1)), moderately high rates of O(2) dissociation ( approximately 5-15 s(-1)), and high oxygen affinity (K(d) or P(50) approximately 50 nM). These properties both facilitate O(2) diffusion to respiring N(2) fixing bacteria and reduce O(2) tension in the root nodules of legumes. The Class 1 plant Hbs show weak hexacoordination (K(HisE7) approximately 2), moderate rates of O(2) binding ( approximately 25 muM(-1) s(-1)), very small rates of O(2) dissociation ( approximately 0.16 s(-1)), and remarkably high O(2) affinities (P(50) approximately 2 nM), suggesting a function involving O(2) and nitric oxide (NO) scavenging. The Class 2 Hbs exhibit strong hexacoordination (K(HisE7) approximately 100), low rates of O(2) binding ( approximately 1 muM(-1) s(-1)), moderately low O(2) dissociation rate constants ( approximately 1 s(-1)), and moderate, Mb-like O(2) affinities (P(50) approximately 340 nM), perhaps suggesting a sensing role for sustained low, micromolar levels of oxygen.

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Nitrosyl hydride, HNO or nitroxyl, is the one-electron reduced and protonated form of nitric oxide. HNO is isoelectronic to singlet O 2 , and we have previously reported that deoxy myoglobin traps free HNO to form a stable adduct. In this report, we demonstrate that oxygen-binding hemoglobins from human, soy and clam also trap HNO to form adducts which are stable over a period of weeks. The same species can be formed in higher yield by careful reduction of the ferrous nitrosyl adducts of the proteins. Like the analogous O 2 -Fe II adducts, the HNO adducts are diamagnetic, but with a characteristic HNO resonance in 1 H NMR ca. 15 ppm that splits into doublets for H 15 NO adducts. The 1 H and 15 N NMR resonances, obtained by HSQC experiments, are shown to differentiate subunits and isoforms of proteins within mixtures. An apparent difference in reduction rates of the NO-adducts of the two subunits of human hemoglobin allows assignment of two distinct nitrosyl hydride peaks by a combination of UVvis, NMR and EPR analysis. The two peaks of HNO-hHb have a persistent 3:1 ratio during trapping reactions, demonstrating a kinetic difference between HNO binding at the two subunits. These results show NMR characterization of ferrous HNO adducts as a unique tool sensitive to structural changes within the oxygen-binding cavity, which may be of use in defining modes of oxygen binding in other heme proteins and enzymes. KeywordsHNO; nitroxyl; nitrosyl hydride; dioxygen; globins; heme oxygenase Nitrosyl hydride (HNO), the protonated form of nitroxyl anion (NO − ), has distinct physicochemical properties from its congener nitric oxide (NO), much of which has been defined only recently. 1,2 The anionic form is isoelectronic with dioxygen and exists as a triplet, 3 NO − above pH 12; at lower pH the singlet 1 HNO dominates, but is susceptible to rapid † This research was supported by the National Science Foundation (PJF CHE-0100774) and the National Institutes of Health (PJF 1R21ES016441-01).*To whom correspondence should be addressed. E-mail: pfarmer@uci.edu. . SUPPORTING INFORMATION AVAILABLE. Experimental details available include descriptions of peak fitting for isoforms of native legHb mixtures, timecourse UVvis spectra during the formation of HNO-hHb, EPR characterization of ferrous NO adduct impurities in HNO adduct samples and data for initial rate analysis of HNO trapping by deoxy Mb and hHb. This material is available free of charge via the Internet at http://pubs.acs.org. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2010 June 9. The rate of this dimerization has been reported as 8 × 10 6 M −1 s −1,5 and thus severely limits the lifetime and concentration of HNO generated in solution.(1)HNO is the simplest analogue of alkylnitroso compounds, RNO, long known to bind to ferrous heme proteins. 6 Mansuy and coworkers were the first to describe the binding of RNO compounds to ferrous globins myoglobin (Mb) and human hemoglobin (hHb), 7 as well as to make the analogy of RNO bindi...

Sperm whale myoglobin (Mb) and soybean leghemoglobin (Lba) are two small, monomeric hemoglobins that share a common globin fold but differ widely in many other aspects. Lba has a much higher affinity for most ligands, and the two proteins use different distal and proximal heme pocket regulatory mechanisms to control ligand binding. Removal of the constraint provided by covalent attachment of the proximal histidine to the F-helices of these proteins decreases oxygen affinity in Lba and increases oxygen affinity in Mb, mainly because of changes in oxygen dissociation rate constants. Hence, Mb and Lba use covalent constraints in opposite ways to regulate ligand binding. Swapping the F-helices of the two proteins brings about similar effects, highlighting the importance of this helix in proximal heme pocket regulation of ligand binding. The F7 residue in Mb is capable of weaving a hydrogen-bonding network that holds the proximal histidine in a fixed orientation. On the contrary, the F7 residue in Lba lacks this property and allows the proximal histidine to assume a conformation favorable for higher ligand binding affinity. Geminate recombination studies indicate that heme iron reactivity on picosecond timescales is not the dominant cause for the effects observed in each mutation. Results also indicate that in Lba the proximal and distal pocket mutations probably influence ligand binding independently. These results are discussed in the context of current hypotheses for proximal heme pocket structure and function.

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