The catalytic mechanism for NO production by the mitochondrial enzyme, sulfite oxidase

2019 ◽  
Vol 476 (13) ◽  
pp. 1955-1956
Author(s):  
Bulent Mutus
Keyword(s):  
Steady State ◽  
Cytochrome C ◽  
Protein Engineering ◽  
Catalytic Mechanism ◽  
Spectroscopic Studies ◽  
Sulfite Oxidase ◽  
Nitrite Reduction ◽  
No Production ◽  
Intramolecular Electron Transfer ◽  
Vis Spectroscopy

Abstract Recently, Guenter Schwarz and colleagues published an elegant study in the Biochemical Journal (2019) 476, 1805–1815 which combines kinetic and spectroscopic studies with protein engineering to provide a mechanism for sulfite oxidase (SO)-catalyzed nitrite reduction that yields nitric oxide (NO). This work is noteworthy as it demonstrates that (i) for NO generation, both sulfite and nitrite must bind to the same molybdenum (Mo) center; (ii) upon sulfite reduction, Mo is reduced from +6 (MoVI) to +4 (MoIV) and MoIV reduces nitrite to NO yielding MoV; (iii) the heme moiety, linked to the Mo-center by an 11 amino acid residue tether, gets reduced by intramolecular electron transfer (IET) resulting in MoV being oxidized to MoVI; (iv) the reduced heme transfers its electron to a second nitrite molecule converting it to NO; (v) the authors demonstrate steady-state NO production in the presence of the natural electron acceptor cytochrome c; (vi) Finally, the authors use protein engineering to shorten the heme tether to reduce the heme-Mo-center distance with the aim of increasing NO production. Consequently, the rate of IET to cytochrome c is decreased but the enzymatic turnover rate for NO production is increased by ∼10-fold. This paper is unique as it provides strong evidence for a novel mechanism for steady-state NO production for human mitochondrial SO and serves as a potential template for studying NO production mechanisms in other enzymes by integrating the information gained from enzyme kinetics with EPR and UV/vis spectroscopy and protein engineering.

Mechanism of nitrite-dependent NO synthesis by human sulfite oxidase

2019 ◽  
Vol 476 (12) ◽  
pp. 1805-1815 ◽  
Author(s):  
Daniel Bender ◽  
Alexander Tobias Kaczmarek ◽  
Dimitri Niks ◽  
Russ Hille ◽  
Guenter Schwarz
Keyword(s):  
Electron Transfer ◽  
Spectroscopic Studies ◽  
Sulfite Oxidase ◽  
Nitrite Reduction ◽  
Intramolecular Electron Transfer ◽  
Intermembrane Space ◽  
Electron Transfer Mechanism ◽  
Physiological Relevance ◽  
Order Of Magnitude ◽  
Human Sulfite Oxidase

AbstractIn addition to nitric oxide (NO) synthases, molybdenum-dependent enzymes have been reported to reduce nitrite to produce NO. Here, we report the stoichiometric reduction in nitrite to NO by human sulfite oxidase (SO), a mitochondrial intermembrane space enzyme primarily involved in cysteine catabolism. Kinetic and spectroscopic studies provide evidence for direct nitrite coordination at the molybdenum center followed by an inner shell electron transfer mechanism. In the presence of the physiological electron acceptor cytochrome c, we were able to close the catalytic cycle of sulfite-dependent nitrite reduction thus leading to steady-state NO synthesis, a finding that strongly supports a physiological relevance of SO-dependent NO formation. By engineering SO variants with reduced intramolecular electron transfer rate, we were able to increase NO generation efficacy by one order of magnitude, providing a mechanistic tool to tune NO synthesis by SO.

Intramolecular electron transfer controls nitrite reduction in molybdenum-containing sulfite oxidase

Nitric Oxide ◽  
2014 ◽  
Vol 42 ◽  
pp. 113
Author(s):  
Guenter Schwarz ◽  
Sabina Krizowski ◽  
Jun Wang ◽  
Dimitri Niks ◽  
Courtney Sparacino-Watkins ◽  
...  
Keyword(s):  
Electron Transfer ◽  
Sulfite Oxidase ◽  
Nitrite Reduction ◽  
Intramolecular Electron Transfer

Routes of electron transfer in beef heart cytochrome c oxidase: is there a unique pathway used by all reductants?

1992 ◽  
Vol 70 (5) ◽  
pp. 301-308 ◽  
Author(s):  
M. Crinson ◽  
P. Nicholls
Keyword(s):  
Electron Transfer ◽  
Steady State ◽  
Cytochrome C ◽  
Cytochrome C Oxidase ◽  
Reduction Rate ◽  
Artificial Substrates ◽  
Intramolecular Electron Transfer ◽  
State Reduction ◽  
Hydrogen Donors ◽  
Slow Turnover

Cytochrome c oxidase oxidizes several hydrogen donors, including TMPD (N,N,N′,N′-tetramethyl-p-phenylenediamine) and DMPT (2-amino-6,7-dimethyl-5,6,7,8-tetrahydropterine), in the absence of the physiological substrate cytochrome c. Maximal enzyme turnovers with TMPD and DMPT alone are rather less than with cytochrome c, but much greater than previously reported if extrapolated to high reductant levels and (or) to 100% reduction of cytochrome a in the steady state. The presence of cytochrome c is, therefore, not necessary for substantial intramolecular electron transfer to occur in the oxidase. A direct bimolecular reduction of cytochrome a by TMPD is sufficient to account for the turnover of the enzyme. CuA may not be an essential component of the TMPD oxidase pathway. DMPT oxidation seems to occur more rapidly than the DMPT – cytochrome a reduction rate and may therefore imply mediation of CuA. Both "resting" and "pulsed" oxidases contain rapid-turnover and slow-turnover species, as determined by aerobic steady-state reduction of cytochrome a by TMPD. Only the "rapid" fraction (≈70% of the total with resting and ≈85% of the total with pulsed) is involved in turnover. We conclude that electron transfer to the a3CuB binuclear centre can occur either from cytochrome a or CuA, depending upon the redox state of the binuclear centre. Under steady-state conditions, cytochrome a and CuA may not always be in rapid equilibrium. Rapid enzyme turnover by either natural or artificial substrates may require reduction of both and two pathways of electron transfer to the a3CuB centre.Key words: cytochrome c oxidase, cytochrome a, respiration, cyanide, stopped flow.

scholarly journals The importance of the interdomain hinge in intramolecular electron transfer in flavocytochrome b2

1993 ◽  
Vol 291 (1) ◽  
pp. 89-94 ◽  
Author(s):  
P White ◽  
F D C Manson ◽  
C E Brunt ◽  
S K Chapman ◽  
G A Reid
Keyword(s):  
Electron Transfer ◽  
Steady State ◽  
Cytochrome C ◽  
Kinetic Properties ◽  
Stopped Flow ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Cytochrome C Reductase ◽  
Flavocytochrome B2 ◽  
Cytochrome C Reduction

The two distinct domains of flavocytochrome b2 (L-lactate:cytochrome c oxidoreductase) are connected by a typical hinge peptide. The amino acid sequence of this interdomain hinge is dramatically different in flavocytochromes b2 from Saccharomyces cerevisiae and Hansenula anomala. This difference in the hinge is believed to contribute to the difference in kinetic properties between the two enzymes. To probe the importance of the hinge, an interspecies hybrid enzyme has been constructed comprising the bulk of the S. cerevisiae enzyme but containing the H. anomala flavocytochrome b2 hinge. The kinetic properties of this ‘hinge-swap’ enzyme have been investigated by steady-state and stopped-flow methods. The hinge-swap enzyme remains a good lactate dehydrogenase as is evident from steady-state experiments with ferricyanide as acceptor (only 3-fold less active than wild-type enzyme) and stopped-flow experiments monitoring flavin reduction (2.5-fold slower than in wild-type enzyme). The major effect of the hinge-swap mutation is to lower dramatically the enzyme's effectiveness as a cytochrome c reductase; kcat. for cytochrome c reduction falls by more than 100-fold, from 207 +/- 10 s-1 (25 degrees C, pH 7.5) in the wild-type enzyme to 1.62 +/- 0.41 s-1 in the mutant enzyme. This fall in cytochrome c reductase activity results from poor interdomain electron transfer between the FMN and haem groups. This can be demonstrated by the fact that the kcat. for haem reduction in the hinge-swap enzyme (measured by the stopped-flow method) has a value of 1.61 +/- 0.42 s-1, identical with the value for cytochrome c reduction and some 300-fold lower than the value for the wild-type enzyme. From these and other kinetic parameters, including kinetic isotope effects with [2-2H]lactate, we conclude that the hinge plays a crucial role in allowing efficient electron transfer between the two domains of flavocytochrome b2.

scholarly journals Increased nitrite reductase activity of fetal versus adult ovine hemoglobin

2009 ◽  
Vol 296 (2) ◽  
pp. H237-H246 ◽  
Author(s):  
Arlin B. Blood ◽  
Mauro Tiso ◽  
Shilpa T. Verma ◽  
Jennifer Lo ◽  
Mahesh S. Joshi ◽  
...  
Keyword(s):  
Nitrite Reductase ◽  
Chronic Hypoxia ◽  
Reductase Activity ◽  
Fetal Hemoglobin ◽  
Nitrite Reduction ◽  
No Production ◽  
Low Oxygen ◽  
Nitrite Reductase Activity ◽  
Adult Hemoglobin ◽  
Severe Ischemia

Growing evidence indicates that nitrite, NO2−, serves as a circulating reservoir of nitric oxide (NO) bioactivity that is activated during physiological and pathological hypoxia. One of the intravascular mechanisms for nitrite conversion to NO is a chemical nitrite reductase activity of deoxyhemoglobin. The rate of NO production from this reaction is increased when hemoglobin is in the R conformation. Because the mammalian fetus exists in a low-oxygen environment compared with the adult and is exposed to episodes of severe ischemia during the normal birthing process, and because fetal hemoglobin assumes the R conformation more readily than adult hemoglobin, we hypothesized that nitrite reduction to NO may be enhanced in the fetal circulation. We found that the reaction was faster for fetal than maternal hemoglobin or blood and that the reactions were fastest at 50–80% oxygen saturation, consistent with an R-state catalysis that is predominant for fetal hemoglobin. Nitrite concentrations were similar in blood taken from chronically instrumented normoxic ewes and their fetuses but were elevated in response to chronic hypoxia. The findings suggest an augmented nitrite reductase activity of fetal hemoglobin and that the production of nitrite may participate in the regulation of vascular NO homeostasis in the fetus.

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