Reactivity of glass-embedded met hemoglobin derivatives toward external NO: implications for nitrite-mediated production of bioactive NO

Abstract

Many protein reactions are exceedingly difficult to dissect under standard conditions due to low concentrations of reactants and intermediates. A case in point are several proposed reactions of hemoglobin with both nitrite and nitric oxide. In the present work, glassy matrices are used to dynamically control the rate at which externally introduced gaseous NO accesses and reacts with several different met Hb derivatives including the nitrite, nitrate, and aquomet forms. This novel yet general approach reveals a clear difference between nitrite and other ligands including nitrate, water, and an internal imidazole. For nitrate, water, and the internal distal imidazole, the observed spectral changes indicate that NO entering the distal heme pocket is effective in displacing these ligands from the ferric heme iron. In contrast, when the ligand is nitrite, the resulting initial spectra indicate the formation of an intermediate that has distinctly ferrous-like properties. The spectrum and the response of DAF fluorescence to the presence of the intermediate are consistent with a recently proposed nitrite anhydrase reaction. This proposed intermediate is especially significant in that it represents a pathway for a nitrite-dependent catalytic process whereby Hb generates relatively long-lived bioactive forms of NO such as S-nitrosoglutathione. The failure to form this intermediate either at low pH or when the glass is extensively dried is consistent with the requirement for a specific conformation of reactants and residue side chains within the distal heme pocket.

Figure 1

Diagram of the protocol used for exposing thin Hb containing glassy films to gaseous nitric oxide (gNO).

Figure 2

Q band absorption spectrum of three different glass-embedded met Hbs as a function of time before and subsequent to the addition of gNO at time t=0 minutes. The labeled dotted reference lines indicate the peak positions for the NO metHb derivative (NO-Fe³⁺Hb). Unless other wise noted all samples are prepared from a stock solution at pH 7.5.

Figure 3

Q band absorption spectrum of glass-embedded aquomet Hb (Panel a) and nitrite metHb (Panel b) as a function of time before and subsequent to the addition of gNO at time t=0 minutes. The pairs of dotted lines at 535 and 565 nm, 543 and 573 nm, 538 and 567 nm indicate the approximate peak positions for NO metHb, NO ferrousHb and nitrite metHb. These glassy samples were allowed to dry for a longer period than for the samples associated with Fig. 2.

Figure 4

The pH dependence of the Q band absorption spectrum of glass-embedded nitrite metHb as a function of time before and subsequent to the addition of gNO at time t=0 minutes. Panels a and b correspond to samples prepared at pH 6.5 and 7.5, respectively.

Figure 5

The Q band absorption spectrum of glass-embedded nitrite metHb (Panels a and b) and nitrate metHb (Panel c) as a function of time before and subsequent to the addition of gNO at time t=0 minutes. The samples giving rise to the spectra shown in Panels b and c were dried for a much longer period than the sample associated with Panel a.

Figure 6

The Q band absorption spectrum of a very dry glass-embedded nitrite metHb sample as a function of time before and subsequent to the addition of gNO at time t=0 minutes. The dotted lines show the peak positions for NO metHb.

Figure 7

The Q band absorption spectrum of a very dry glass-embedded aquometHb sample as a function of time before and subsequent to the addition of gNO at time t=0 minutes. With the extreme drying the aquomet Hb spectrum evolved to the one shown which is characteristic of the Hb hemichrome (see text for details).

Figure 8

Increase in fluorescence from an anaerobic DAF-2 solution (10 μM, pH 7.5) exposed to glassy film containing a detectable level of “intermediate” as reflected in the absorption spectrum. The intermediate is accessed by exposing a thin glassy sample of the nitrite metHb derivative to gaseous NO under anaerobic conditions until the absorption spectrum shows indications of formation of the intermediate (see inset). The NO is then thoroughly flushed out and the glassy sample still under rigorous anaerobic conditions is then exposed to an aliquot of solution containing DAF-2 (see text for detains). The control spectrum shown as the bottom fluorescence trace is from the DAF-2 solution prior to exposure to the glassy film and the sequence showing the increase in intensity is the progression as a function of time after the DAF-2 solution is exposed to the glassy film (see text for details). Inset shows the initial absorption spectrum of the met Hb nitrite sample prior to exposure to gaseous NO under anaerobic conditions (in red) and the spectrum of the “intermediate” that occurs hours after the sample is exposed to NO under anaerobic conditions (in black). Not shown is the lack of response of the DAF fluorescence when, under anaerobic conditions, the DAF solution is exposed to: NO by itself, the starting glassy sample prior to introducing NO, and the sample after fully evolving to the ferrous NOHb derivative.